Barriers for facilitating biological reactions

ABSTRACT

The present invention relates to systems, devices, and methods for performing biological reactions. In particular, the present invention relates to the use of lipophilic, water immiscible, or hydrophobic barriers in sample separation, purification, modification, and analysis processes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/395,020 filed Feb. 27, 2009, now pending, which claims priority toU.S. Provisional Patent Application Ser. No. 61/032,655, filed Feb. 29,2008, each of which are herein incorporated by reference in theirentirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant NoCCF0329957 awarded by the National Science Foundation and Grant No.5R01EB001418-03 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to systems, devices, and methods forperforming biological reactions. In particular, the present inventionrelates to the use of hydrophobic, water-immiscible, or lipophilicbarriers in sample separation, purification, modification, and analysisprocesses.

BACKGROUND OF THE INVENTION

There is a great need for cost-effective, easy to use systems, methods,and devices for analyzing biological samples. Many commerciallyavailable systems cost tens to hundreds of thousands of dollars and havemany moving parts which make them prone to failure. Because of the costand complexity of such systems, their use has generally been limited toclinical laboratories which have the personnel and services needed tosupport their operation and maintenance.

One class of fully integrated automated analyzers, represented by theAbbott Architect, Siemens Centaur, Roche Elecsys, and others, performimmunoassays. Another class of modular analyzers, represented by theAbbott m2000, Roche COBAS, bioMérieux NucliSENS and others, performnucleic acid assays. Much of the complexity of these systems is a resultof separation steps involved in processing the assays.

Modular systems are also frequently used in research laboratories.Immunoassay separations may be performed by plate washers such asTitertek MAP-C2, BioTek ELx50, Tecan PW 96/384 and others. Nucleic acidseparations are performed by systems such as the Applied BiosystemsPRISM™ 6100, Invitrogen iPrep, Thermo Scientific KingFisher, PromegaMaxwell, and others.

The availability of low-cost, reliable analyzers is of particularconcern as it relates to the diagnosis and management of disease aroundthe world. This problem is vividly illustrated by the problemsassociated with management of HIV infections. Many technologies existthat permit detection of nucleic acids or protein levels associated withHIV. This detection is important for managing the patient care of thoseinfected by HIV. However, the cost and complexity of these systemsprohibits their widespread use.

SUMMARY OF THE INVENTION

The present invention relates to systems, devices, and methods forperforming biological reactions. In particular, the present inventionrelates to the use of hydrophobic, water or alcohol-immiscible, orlipophilic barriers in sample separation, purification, modification,and analysis processes.

In some embodiments, the present invention provides a biological samplepurification and/or analysis device, comprising: a plurality of sampleprocessing chambers comprising reagents for biological molecule or cellpurification, modification, analysis, and/or detection; and a lipophilicsubstance in between (e.g., separating) two or more of the chambers. Insome embodiments, the lipophilic substance is a wax. In someembodiments, the wax is a phase change wax that can take liquid or solidforms of pre-determined temperatures. For example, in some embodiments,the wax takes solid form at storing or shipping temperatures and liquidform at reaction temperature (e.g., room temperature). In someembodiments, the lipophilic substance is an oil. In some embodiments,there are two reaction chambers. In some embodiments, there are threereaction chambers. In some embodiments, there are four reactionchambers. In some embodiments, there are five reactions chambers. Insome embodiments, there are six or more reaction chambers (e.g., 7, 8,9, 10, 11, . . . , 20, . . . ). In some embodiments, the lipophilicmaterial provides a contiguous barrier between two or more of thechambers (i.e., a sample passes from a first chamber directly into thelipophilic material and directly out of the lipophilic material into thesecond chamber). In other embodiments, there is air, liquid, or othermaterial between the lipophilic material and one or more of thechambers. In such embodiments, the lipophilic material is positionedsuch that a sample or biological molecule to be processed passes throughthe lipophilic material at some point between its transit from a firstchamber to a second chamber.

In some embodiments, all of, or a subset of the reaction chambers arededicated for sample purification. For example, one or more reactionchambers contain reagents that cause a sample purification event tooccur, including, but not limited to, cell lysis, biological moleculecapture, biological molecule separation, and cellular culture,purification, and/or analysis. In some embodiments, all of, or a subsetof the reactions chambers are dedicated for sample modification. Forexample, one or more reaction chambers contain reagents that cause abiological molecule (e.g., nucleic acid, protein, lipid, etc.) or cellmodification event to occur, including, but not limited to,amplification, ligation, cleavage, labeling, extension, degradation,association with a ligand, oligomerization, transfection,transformation, transgenesis, division, differentiation, and the like.In some embodiments, all of, or a subset of the reaction chambers arededicated for sample analysis. For example, one or more reactionchambers contain reagents or other components that permit detection orother types of analysis of a biological molecule or cells of interest.In some embodiments, the chambers contain reagents that permitdevelopment of a color, fluorescent signal, luminescent signal or otherdetectable characteristic. In some embodiments, the chambers areconfigured to optimize signal detection by a signal reader (e.g., colorreader, fluorescence reader, luminescence reader, the human eye, etc.).One or more of the chambers may be used for multiple different tasks,including purification, modification, analysis, and/or detection.

The present invention is not limited by the manner in which the chambersare configured or separated from one another. The chambers may each bethe same size and shape as one another or may be different sizes orshapes. A wide variety of configurations may be used. In someembodiments, the chambers are wells and the lipophilic barrier sits ontop of or below the wells, such that any material that is transferredfrom one chamber to another, must pass through the lipophilic materialby moving up or down, and over. In some embodiments, the chambers arecreated by the existence of the lipophilic material. For example, insome embodiments, a lipophilic material is deposited along one or morepoints in a channel or channels (e.g., in a glass, plastic, or ceramictube), to create barriers between zones in the channel or channels. Thechannel may be any size, including small sizes such as capillary tubesor microfluidic channels. In some embodiments, that chambers andbarriers are configured so that a sample or biological molecule ofinterest must travel through a linear series of reaction chambers.However, in other embodiments, a sample or biological molecule in afirst reaction chamber may optionally skip one or more other chambers.In some embodiments, the chambers are housed in a device that has a sizeor shape configured to fit existing laboratory equipment (e.g.,automated robotic arms, plate holders (e.g., 96-well), thermocyclers,fluorescent detectors, etc.). In some embodiments, channels are used toseparate reaction chambers, where all or a portion of the channelcontains the lipophilic material. For example, in some embodiments, thedevice is configured similar to a 96-well or 384-well plate withchannels connecting two more of the wells. In some embodiments, apathway between two chambers contains air, water, or other fluids, wherethe sample passes through the air, water, or other fluid before and/orafter entering or leaving the lipophilic material.

In some embodiments, reaction chambers are microwells or microtubescontaining hydrophilic solutions and the lipophilic substance is placedon top of or below the solution in a subset of the chambers or in aseparate chamber.

In some embodiments, the device comprises a transport mechanism thatpermits transfer of a desired material from one reaction chamber toanother through the lipophilic material. For example, in someembodiments, a biological molecule of interest is associated with amagnetic particle, such as a magnetic bead, in one of the reactionchambers. The biological molecule of interest is moved from one chamberto the other by application of a magnetic field (e.g., from a magnet)that causes the magnetic particle to travel from a first chamber,through the lipophilic barrier, to a second chamber. In otherembodiments, an electrical field is created to move biological moleculesor components associated with the biological molecules (e.g., ligands,beads, charge tags, etc.), using charge, from one reaction chamber toanother. In some embodiments, centrifugal force is used to movebiological molecules of interest from one chamber to another, throughthe lipophilic barrier. In some embodiments, pressure from a vacuum orfrom suction is used to move materials between chambers. The presentinvention is not limited by the mechanism of transport.

In some embodiments, the device comprises a vapor barrier to prevent orreduce the loss of liquid during handling or use. In some embodiments,the device is composed of a plurality of thin layers of material stackedon one another. For example, in some embodiments, the layers comprise analuminum foil layer sandwiched between plastic layers.

In some embodiments, the devices of the invention are provided as asystem (e.g., kit) that includes one or more other components thatpermit sample acquisition, sample handling, sample disposal, datacollection, data analysis, and data presentation. These components maybe separate devices or may be integrated into a single multi-componentdevice. These components may include, but are not limited to, medicaldevices, environmental sample handling devices, protein purificationdevices, nucleic acid purification devices, computers, software, and thelike. One or more components of the system or device can be automated.In some embodiments, one or more components of the system are configuredto work without automation. For example, in a non-automated system, ahandheld magnet is provided to move samples from one chamber to another,a heat block or water bath is used to create the desired reactionstemperatures, a hand held fluorescence detector is used to detect signalor a signal is observed by eye.

The systems and devices of the invention find use with a wide variety ofsamples. For example, in some embodiments, a sample is a biological orenvironmental sample. Biological samples may be obtained from animals(including humans) and encompass fluids, solids, tissues, and gases.Biological samples include blood products, such as plasma, serum and thelike, as well as cerebrospinal fluid, sputum, bronchial washing,bronchial aspirates, urine, lymph fluids, and various externalsecretions of the respiratory, intestinal and genitourinary tracts,tears, saliva, milk, white blood cells, myelomas, biological fluids suchas cell culture supernatants, tissue (fixed or not fixed), cell (fixedor not fixed), and the like. Environmental samples include, but are notlimited to, environmental material such as surface matter, soil, water,and industrial samples.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a cartridge for sample purification and PCR in accord withsome embodiments of the present invention.

FIG. 2 shows a cartridge for sample purification of some embodiments ofthe present invention.

FIG. 3 shows layers of a foil laminate used in constructing cartridgesof some embodiments of the present invention.

FIG. 4 shows a drawing of a foil laminate cartridge used in someembodiments of the present invention.

FIG. 5 shows (a) the position of a permanent magnet with respect to twoimmiscible fluids and (b) a surface plot of magnetic force on a particlein the x and y directions.

FIG. 6 shows an experimental set up for estimation of surface tensionusing the weight drop method.

FIG. 7 shows an illustration of the various stages of a sandwich assayin a tube-based microfluidic system.

FIG. 8 shows a plot of FL1 height verses Log (Biotin concentration).

FIG. 9 shows a plot of events recorded by the flow cytometer versesforward scatter, side scatter and FL1 height.

FIG. 10 shows an illustration of the movement of streptavidin coatedmagnetic particles from one capillary to another followed by reactionwith biotin.

FIG. 11 a shows the signal of streptavidin coated particles after movingthrough oil containing a fluorescent dye. FIG. 11 b shows the signal ofstreptavidin coated particles after moving through oil containing afluorescent dye followed by agitation of the particles in PBS buffer.

FIG. 12 shows a schematic of a two-chamber cuvette used in someembodiments of the present invention.

FIG. 13 shows a schematic of a two-chamber cuvette used in someembodiments of the present invention.

FIG. 14 shows qRT-PCR for HIV-1 from plasma using an immiscible phasefilter (IPF) method: Standard curve of C_(t) values for 4 different RNAconcentrations run in duplicate plotted verses the log₁₀ of the HIV-1viral copy number.

FIG. 15 shows a Bland-Altman plot comparing the IPF and manual method ofpurification: Solid black squares show difference between the twomethods, solid line (y=−0.00772) plots the mean difference between thetwo methods and the dashes lines show the mean+2 and −2 standarddeviations (SD) of the mean.

FIG. 16 shows IPF quantification by qPCR of Chlamydia from urinesamples.

FIG. 17 shows IPF quantification by qPCR of gonorrhea from urinesamples.

FIG. 18 shows Bland-Altman plot comparing the IPF and manual method ofpurification of Chlamydia.

FIG. 19 shows a Bland-Altman plot comparing the IPF and manual method ofpurification of gonorrhea.

FIG. 20 shows IPF PCR for proviral DNA from 25 μL WB.

FIG. 21 shows a Bland-Altman plot comparing the IPF and manual method ofpurification.

DEFINITIONS

To facilitate an understanding of this disclosure, terms are definedbelow:

As used herein, the term “lipophilic material” refers to any substancewhich is substantially immiscible in water, alcohol, or otherhydrophilic or aqueous fluid. In some embodiments, lipophilic materialsof the present invention have a low solubility for substances thatinterfere with a particular biological process such as nucleicamplification or biomolecule detection. In some embodiments, lipophilicmaterials of the invention have a low vapor pressure. Lipophilicsubstances tend to interact within themselves and with other substancesthrough van der Waals forces. They have little to no capacity to formhydrogen bonds. Lipophilic substances typically have large o/w(oil/water) partition coefficients.

“Water insoluble” and “hydrophobic” materials are used synonymously inthis specification. The terms include polymers that are practicallyinsoluble in water and freely soluble in volatile lipophilic solventssuch as methylene chloride and non-volatile hydrophilic solvents,particularly N-methylpyrollidone (NMP).

“Water-miscible” or “hydrophilic” materials refer to an organic liquidthat can be diluted with at least an equal part of water withoutseparation.

A property of “water-immiscible” or “lipophilic” materials is that theycannot be diluted with at least an equal part of water withoutseparation.

“Purified polypeptide” or “purified protein” or “purified nucleic acid”means a polypeptide or nucleic acid of interest or fragment thereofwhich is essentially free of, e.g., contains less than about 50%,preferably less than about 70%, and more preferably less than about 90%,cellular components with which the polypeptide or polynucleocide ofinterest is naturally associated.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, which is separated from some orall of the coexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat the vector or composition is not part of its natural environment.

“Polypeptide” and “protein” are used interchangeably herein and includeall polypeptides as described below. The basic structure of polypeptidesis well known and has been described in innumerable textbooks and otherpublications in the art. In this context, the term is used herein torefer to any peptide or protein comprising two or more amino acidsjoined to each other in a linear chain by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types.

It will be appreciated that polypeptides often contain amino acids otherthan the 20 amino acids commonly referred to as the 20 naturallyoccurring amino acids, and that many amino acids, including the terminalamino acids, may be modified in a given polypeptide, either by naturalprocesses, such as processing and other post-translationalmodifications, but also by chemical modification techniques which arewell known to the art. Even the common modifications that occurnaturally in polypeptides are too numerous to list exhaustively here,but they are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature, and they arewell known to those of skill in the art. Among the known modificationswhich may be present in polypeptides of the present are, to name anillustrative few, acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid of lipid derivative, covalent attachment ofphosphatidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myrisoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination.

Such modifications are well known to those of skill and have beendescribed in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as for instance Proteins—Structure and Molecular Properties,2.sup.nd Ed., T. E. Creighton, W. H. Freeman and Company, New York(1993). Many detailed reviews are available on this subject, such as,for example, those provided by Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pg. 1-12 in PosttranslationalCovalent Modification of Proteins, B. C. Johnson, Ed., Academic Press,New York (1983); Seifter et al., Analysis for protein modifications andnonprotein cofactors, Meth. Enzymol. 182: 626-646 (1990) and Rattan etal., Protein synthesis: Posttranslational Modifications and Aging, AnnN.Y. Acad. Sci. 663: 48-62 (1992).

It will be appreciated, as is well known and as noted above, thatpolypeptides are not always entirely linear. For instance, polypeptidesmay be branched as a result of ubiquitination, and they may be circular,with or without branching, generally as a result of posttranslationalevents, including natural processing events and events brought about byhuman manipulation which do not occur naturally. Circular, branched, andbranched circular polypeptides may be synthesized by non-translationalnatural process and by entirely synthetic methods as well.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring andsynthetic polypeptides. For instance, the amino terminal residue ofpolypeptides made in E. coli, prior to proteolytic processing, almostinvariably will be N-formylmethionine.

The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcells often carry out the same posttranslational glycosylations asmammalian cells, and, for this reason, insect cell expression systemshave been developed to express efficiently mammalian proteins havingnative patterns of glycosylation. Similar considerations apply to othermodifications.

It will be appreciated that the same type of modification may be presentin the same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.

In general, as used herein, the term polypeptide encompasses all suchmodifications, particularly those that are present in polypeptidessynthesized by expressing a polynucleotide in a host cell.

The term “mature” polypeptide refers to a polypeptide which hasundergone a complete, post-translational modification appropriate forthe subject polypeptide and the cell of origin.

A “fragment” of a specified polypeptide refers to an amino acid sequencewhich comprises at least about 3-5 amino acids, more preferably at leastabout 8-10 amino acids, and even more preferably at least about 15-20amino acids derived from the specified polypeptide.

The term “immunologically identifiable with/as” refers to the presenceof epitope(s) and polypeptide(s) which also are present in and areunique to the designated polypeptide(s). Immunological identity may bedetermined by antibody binding and/or competition in binding. Theuniqueness of an epitope also can be determined by computer searches ofknown data banks, such as GenBank, for the polynucleotide sequence whichencodes the epitope and by amino acid sequence comparisons with otherknown proteins.

As used herein, “epitope” means an antigenic determinant of apolypeptide or protein. Conceivably, an epitope can comprise three aminoacids in a spatial conformation which is unique to the epitope.Generally, an epitope consists of at least five such amino acids andmore usually, it consists of at least eight to ten amino acids. Methodsof examining spatial conformation are known in the art and include, forexample, x-ray crystallography and two-dimensional nuclear magneticresonance.

A “conformational epitope” is an epitope that is comprised of a specificjuxtaposition of amino acids in an immunologically recognizablestructure, such amino acids being present on the same polypeptide in acontiguous or non-contiguous order or present on different polypeptides.

A polypeptide is “immunologically reactive” with an antibody when itbinds to an antibody due to antibody recognition of a specific epitopecontained within the polypeptide. Immunological reactivity may bedetermined by antibody binding, more particularly, by the kinetics ofantibody binding, and/or by competition in binding using ascompetitor(s) a known polypeptide(s) containing an epitope against whichthe antibody is directed. The methods for determining whether apolypeptide is immunologically reactive with an antibody are known inthe art.

As used herein, the term “immunogenic polypeptide containing an epitopeof interest” means naturally occurring polypeptides of interest orfragments thereof, as well as polypeptides prepared by other means, forexample, by chemical synthesis or the expression of the polypeptide in arecombinant organism.

“Purified product” refers to a preparation of the product which has beenisolated from the cellular constituents with which the product isnormally associated and from other types of cells which may be presentin the sample of interest.

“Analyte,” as used herein, is the substance to be detected which may bepresent in the test sample, including, biological molecules of interest,small molecules, pathogens, and the like. The analyte can include aprotein, a polypeptide, an amino acid, a nucleotide target and the like.The analyte can be soluble in a body fluid such as blood, blood plasmaor serum, urine or the like. The analyte can be in a tissue, either on acell surface or within a cell. The analyte can be on or in a celldispersed in a body fluid such as blood, urine, breast aspirate, orobtained as a biopsy sample.

A “specific binding member,” as used herein, is a member of a specificbinding pair. That is, two different molecules where one of themolecules, through chemical or physical means, specifically binds to thesecond molecule. Therefore, in addition to antigen and antibody specificbinding pairs of common immunoassays, other specific binding pairs caninclude biotin and avidin, carbohydrates and lectins, complementarynucleotide sequences, effector and receptor molecules, cofactors andenzymes, enzyme inhibitors, and enzymes and the like. Furthermore,specific binding pairs can include members that are analogs of theoriginal specific binding members, for example, an analyte-analog.Immunoreactive specific binding members include antigens, antigenfragments, antibodies and antibody fragments, both monoclonal andpolyclonal and complexes thereof, including those formed by recombinantDNA molecules.

Specific binding members include “specific binding molecules.” A“specific binding molecule” intends any specific binding member,particularly an immunoreactive specific binding member. As such, theterm “specific binding molecule” encompasses antibody molecules(obtained from both polyclonal and monoclonal preparations), as well as,the following: hybrid (chimeric) antibody molecules (see, for example,Winter, et al., Nature 349: 293-299 (1991), and U.S. Pat. No.4,816,567); F(ab′).sub.2 and F(ab) fragments; Fv molecules (non-covalentheterodimers, see, for example, Inbar, et al., Proc. Natl. Acad. Sci.USA 69: 2659-2662 (1972), and Ehrlich, et al., Biochem. 19: 4091-4096(1980)); single chain Fv molecules (sFv) (see, for example, Huston, etal., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)); humanizedantibody molecules (see, for example, Riechmann, et al., Nature 332:323-327 (1988), Verhoeyan, et al., Science 239: 1534-1536 (1988), and UKPatent Publication No. GB 2,276,169, published 21 Sep. 1994); and, anyfunctional fragments obtained from such molecules, wherein suchfragments retain immunological binding properties of the parent antibodymolecule.

The term “hapten,” as used herein, refers to a partial antigen ornon-protein binding member which is capable of binding to an antibody,but which is not capable of eliciting antibody formation unless coupledto a carrier protein.

A “capture reagent,” as used herein, refers to an unlabeled specificbinding member which is specific either for the analyte as in a sandwichassay, for the indicator reagent or analyte as in a competitive assay,or for an ancillary specific binding member, which itself is specificfor the analyte, as in an indirect assay. The capture reagent can bedirectly or indirectly bound to a solid phase material before theperformance of the assay or during the performance of the assay, therebyenabling the separation of immobilized complexes from the test sample.

The “indicator reagent” comprises a “signal-generating compound”(“label”) which is capable of generating and generates a measurablesignal detectable by external means. In some embodiments, the indicatorreagent is conjugated (“attached”) to a specific binding member. Inaddition to being an antibody member of a specific binding pair, theindicator reagent also can be a member of any specific binding pair,including either hapten-anti-hapten systems such as biotin oranti-biotin, avidin or biotin, a carbohydrate or a lectin, acomplementary nucleotide sequence, an effector or a receptor molecule,an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme andthe like. An immunoreactive specific binding member can be an antibody,an antigen, or an antibody/antigen complex that is capable of bindingeither to the polypeptide of interest as in a sandwich assay, to thecapture reagent as in a competitive assay, or to the ancillary specificbinding member as in an indirect assay. When describing probes and probeassays, the term “reporter molecule” may be used. A reporter moleculecomprises a signal generating compound as described hereinaboveconjugated to a specific binding member of a specific binding pair, suchas carbazole or adamantane.

The various “signal-generating compounds” (labels) contemplated includechromagens, catalysts such as enzymes, luminescent compounds such asfluorescein and rhodamine, chemiluminescent compounds such asdioxetanes, acridiniums, phenanthridiniums and luminol, radioactiveelements and direct visual labels. Examples of enzymes include alkalinephosphatase, horseradish peroxidase, beta-galactosidase and the like.The selection of a particular label is not critical, but it should becapable of producing a signal either by itself or in conjunction withone or more additional substances.

“Solid phases” (“solid supports”) are known to those in the art andinclude the walls of wells of a reaction tray, test tubes, polystyrenebeads, magnetic or non-magnetic beads, nitrocellulose strips, membranes,microparticles such as latex particles, and others. The “solid phase” isnot critical and can be selected by one skilled in the art. Thus, latexparticles, microparticles, magnetic or non-magnetic beads, membranes,plastic tubes, walls of microtiter wells, glass or silicon chips, areall suitable examples. It is contemplated and within the scope of thepresent invention that the solid phase also can comprise any suitableporous material.

As used herein, the terms “detect”, “detecting”, or “detection” maydescribe either the general act of discovering or discerning or thespecific observation of a detectably labeled composition.

The term “polynucleotide” refers to a polymer of ribonucleic acid (RNA),deoxyribonucleic acid (DNA), modified RNA or DNA, or RNA or DNAmimetics. This term, therefore, includes polynucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleoside(backbone) linkages as well as polynucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted polynucleotides are well-known in the art and for thepurposes of the present invention, are referred to as “analogues.”

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule, including but not limited to, DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N-6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-aminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

The term “nucleic acid amplification reagents” includes conventionalreagents employed in amplification reactions and includes, but is notlimited to, one or more enzymes having polymerase activity, enzymecofactors (such as magnesium or nicotinamide adenine dinucleotide(NAD)), salts, buffers, deoxynucleotide triphosphates (dNTPs; forexample, deoxyadenosine triphosphate, deoxyguanosine triphosphate,deoxycytidine triphosphate and deoxythymidine triphosphate) and otherreagents that modulate the activity of the polymerase enzyme or thespecificity of the primers.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides such asan oligonucleotide or a target nucleic acid) related by the base-pairingrules. Complementarity may be “partial,” in which only some of thenucleic acids' bases are matched according to the base pairing rules.Or, there may be “complete” or “total” complementarity between thenucleic acids. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methodswhich depend upon binding between nucleic acids.

The term “homology” refers to a degree of identity. There may be partialhomology or complete homology. A partially identical sequence is onethat is less than 100% identical to another sequence.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the Tm of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein, the term “Tm” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the Tm ofnucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the Tm value may be calculated by theequation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (see e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization (1985). Other referencesinclude more sophisticated computations which take structural as well assequence characteristics into account for the calculation of Tm.

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds, under which nucleic acid hybridizations are conducted. With“high stringency” conditions, nucleic acid base pairing will occur onlybetween nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “weak” or “low”stringency are often required when it is desired that nucleic acidswhich are not completely complementary to one another be hybridized orannealed together.

The term “wild-type” refers to a gene or gene product which has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product which displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally-occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

The term “oligonucleotide” as used herein is defined as a moleculecomprised of two or more deoxyribonucleotides or ribonucleotides,preferably at least 5 nucleotides, more preferably at least about 10-15nucleotides and more preferably at least about 15 to 30 nucleotides, orlonger. The exact size will depend on many factors, which in turndepends on the ultimate function or use of the oligonucleotide. Theoligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof.

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. A first regionalong a nucleic acid strand is said to be upstream of another region ifthe 3′ end of the first region is before the 5′ end of the second regionwhen moving along a strand of nucleic acid in a 5′ to 3′ direction.

When two different, non-overlapping oligonucleotides anneal to differentregions of the same linear complementary nucleic acid sequence, and the3′ end of one oligonucleotide points towards the 5′ end of the other,the former may be called the “upstream” oligonucleotide and the latterthe “downstream” oligonucleotide.

The term “primer” refers to an oligonucleotide which is capable ofacting as a point of initiation of synthesis when placed underconditions in which primer extension is initiated. An oligonucleotide“primer” may occur naturally, as in a purified restriction digest or maybe produced synthetically.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to systems, devices, and methods forperforming biological reactions. In particular, the present inventionrelates to the use of lipophilic barriers in sample separation,purification, modification, and analysis processes.

The systems, devices, and methods of the invention may be configured, ifdesired, as inexpensive and easy-to-use sample purification and/ormodification and/or analysis and/or detection systems. For example,embodiments of the present invention, described herein, provide aneconomical means for widespread biological molecule detection, analysis,and characterization. These systems, device, and methods find many uses.To illustrate aspect and benefits of the invention, its application tonucleic acid and protein analysis, particularly for monitoring HIVinfection and status, are provided below. The invention is not limitedto these illustrative embodiments. In some embodiments, the systems,devices, and methods of the invention are utilized in conjunction withexisting, complex, expensive sample separation, purification,modification, and analysis equipments. For example, in some embodiments,the approaches of the present invention are used for sample preparation(e.g., nucleic acid or polypeptide purification) prior to modificationand/or analysis using traditional equipment (e.g., thermocyclers, massspectrometers, NMR devices, etc.).

There are 35 million adults and children living with HIV/AIDS, 22million of them in sub-Saharan Africa. The average clinic in Africatreats about 400 patients and the problem of transportation is leadingto an increase in the number of clinics rather than the growth of largecentral facilities. Therefore, there is a need for more than 100,000viral load measuring machines. The major limitations of currentlyavailable viral load assays include the cost of the requiredinstruments, the complex and time-consuming procedures leading to theneed for highly trained personnel, and the need for cold-chain shipmentof reagents.

The development of affordable and simple HIV viral load assays is acritical step for improving the quality of AIDS patient care in thedeveloping world. This would require automating complex diagnosticprocedures that are normally performed in a centralized laboratory intosmall point of care (POC) devices; this capability could empowerhealth-care workers and patients with important health-relatedinformation in even the most remote settings. The required HIV viralload assay should preferably deliver answers at the point of care, butmoving it from remote central laboratories to district hospital labscloser to the patient will improve outcomes. Such a device will performseparation, amplification, and detection of HIV with a short turnaroundtime and at an affordable cost. A short time is critical since it wouldreduce the number of machines needed in a clinic and reduce the timespent by the patient at the clinic, thereby reducing the actual cost.

Many challenges must be overcome when conducting HIV viral load assaysboth in centralized laboratories and out in the field. Largelaboratories use automated or semi-automated robotic systems forhigh-volume HIV viral load assays. However, sample processing istypically the most troublesome part of these tests. Currently,sample-processing procedures involve many steps, often requiringcentrifugation and extraction steps. Also, these methods often do notadequately purify the target nucleic acid. They often leave inhibitoryor interfering substances in the reaction mixture that can causeinhibition of the amplification reaction and result in false-negativeresults. The manual nature of current sample-processing techniques alsocan lead to specimen cross-contamination, which can cause false-positiveresults.

Considerable effort has been made in trying to automate the samplepreparation process, since this would allow for the more widespread useof PCR or other nucleic analysis techniques. However, existing automatedhigh-throughput systems perform multiple extraction and purificationsteps, and still require certain manual preparations, including sampleand reagent loading, and waste removal. Hence, highly trainedtechnicians are required to conduct the assay and maintain theinstrument. The automated systems are very expensive because they usecomplex robotic arms to move solutions or magnetic particles andprecision instruments to pipette liquids. The cost of an automatedsystem is often difficult to justify for smaller laboratories,especially those in resource limited settings. Cross-contamination isalso a problem since they employ amplification technologies. Clinicallaboratories often use separate rooms for reagent preparation, samplepreparation, amplification, and post-amplification analysis. For thesereasons, despite the automation, viral load testing is consideredhigh-complexity tests under the Clinical Laboratory ImprovementAmendments (CLIA). To date, no Nucleic Acid Test (NAT) system hasqualified for CLIA-waived status, largely because of the difficulties inautomating sample preparation and reagent handling.

Performing field-use or near-patient NATs involves even more challenges,especially since they will inevitably be conducted by less-experiencedusers in non-laboratory environments. The following systems haverecently been developed for deployment of NATs in the field.

The GeneXpert system by Cepheid (Sunnyvale, Calif.) is one of the firstPCR-based instruments that integrate sample preparation, amplification,and detection. The disposable single-test GeneXpert sample-preparationcartridge consists of four functional components: the cap, the cartridgebody, the valve body assembly, and the micro volume PCR reaction tube.The cartridge body is divided internally into a number of chambers ofvarious sizes and functions, some containing the lyophilized reagentbeads, and each with a port at the bottom for fluidic inflow andoutflow. The chambers are radially arranged around the syringe barrel inthe center. The valve body assembly, located below the cartridge body,is the site of cell lysing and DNA purification, under software control,a rotary valve on the instrument moves the valve body assembly so thatfluids can be aspirated from or dispensed into the appropriate chamberfor mixing, dilution, and washing, according to the programmed assayprotocol. The reaction tube, which projects from the cartridge, receivesthe prepared sample and interfaces with the PCR reactor foramplification and detection of the target analyte. To perform a test onthe GeneXpert system, the operator opens the cartridge cap and loads theliquid sample into the sample chamber. When the operator closes the cap,the cartridge is permanently sealed throughout the testing procedure andbiohazard disposal, eliminating any risk of cross-contamination ofsamples. Cells are lysed by agitating tiny glass beads in the valve bodyassembly by ultrasound generated directly below the cartridge. Theextracted DNA flows into a micro fluidic channel containing immobilizedDNA probes that DNA as the cellular debris flows over. The bound DNA islater released from its attachment site and washed off for PCRamplification.

Another system developed by IQuum Inc (Allston, Mass.) is the LiatMolecular Analyzer based on its proprietary lab-in-a-tube (Liat)technology platform. The Liat tube uses a flexible tube as the samplevessel and contains all assay reagents pre-packed in tube segments. Theunit-dose reagents and internal controls can be held separately in aseries of tube segments in the order they are used for an assay by usingpeelable seals. The peelable seal is formed by a thermal weld of theplastic tube. By applying pressure to the tube segments adjacent to eachseal, the seal can burst open to release reagents. In the Liat analyzer,multiple sample-processor modules are aligned with the Liar tube. Eachmodule consists of an actuator and a clamp, whose positions can becontrolled to manipulate a test sample within a tube. A retractablemagnet is attached to one of the modules for manipulating magneticbeads. When a tube is loaded in the analyzer, the actuators and clampscompress the tube sequentially to move the reagents and controls fromone segment to another. Similarly, by synchronizing the motion of theactuators and clamps, various sample processes can be conducted within atube. Such processes include adjusting a liquid's volume in a segment;releasing a reagent to the adjacent segment; mixing reagents andsamples; agitating and incubating a reaction mixture at a giventemperature; and washing and removing waste from a segment. Waste ismoved toward a waste chamber in the cap while the purified sample movesfurther down the tube. In the lowest chamber, the released DNA isamplified.

Other commercialized real-time PCR devices intended for field useinclude the Ruggedized Advanced Pathogen Identification Device by IdahoTechnology Inc. (Salt Lake City), the Hand-Held Advanced Nucleic AcidAnalyzer by Lawrence Livermore National Laboratory (Livermore, Calif.),and the Bio-Seeq detector by Smiths Detection (Pine Brook, N J).However, these devices do not have automated sample preparation andreagent-handling functions.

The systems mentioned above are a step in the right direction. However,the GeneXpert is still moderately complex to operate and has to beoperated by a trained technician. Since it requires the user to pipetteliquid in the field, a precision measuring instrument would be neededwhich further increases the cost of the system. Although the LiarMolecular Analyzer does not involve measuring precise liquid volumes inthe field, it is expensive because of the complex mechanical systemneeded to move fluids accurately. The Liat tube is difficult tomanufacture, which makes quality control difficult. The tube isdifficult to store and has leakage problems.

The controlled movement and delivery of small quantities of-moleculessuch as proteins and chemical reagents represents an ongoing challengein micro fluidics and is of critical importance for developing POCdevices such as the HIV viral load device. The majority of micro fluidicsystems rely on fluid motion to move solutions of molecules from onelocation to another, and as a result, these systems unnecessarilyconsume solvent and materials and involve complex mechanical systems tocontrol fluid flow. Embodiments of the present invention provide analternative approach to address the problem in the use of magnetic microparticles as carriers to move molecules from one reaction media to thenext and remove all fluid flow from the system. Such an approach wheremagnetic particles are manipulated using magnetic forces allows one tocarry out complex chemical reaction such as viral load testing is aclosed cartridge at a low cost. Since the driving force in the reactionis a magnetic field, the system can be automated, allowing for theconstruction of a portable, reliable instrument for viral load testswith no contamination problems. While the current system is exemplifiedfor sample purification of HIV viral RNA, the platform can easily beextended to other nucleic acid tests and immunoassays, as well asdetection of other biological molecules or non-biological molecule ofinterest.

Embodiments of the present invention provide devices used to performsample purification and analysis assays in a single instrument. In oneexemplary embodiment (See e.g., Example 1) it involves the use ofmagnetic particles as a solid phase for capture of RNA, subsequentpurification and release of RNA to carry out amplification anddetection.

Conventional devices conduct assays by exchanging solutions contactingthe solid phase. The solid phase may be a micro titer well, a microparticle, or packed column. Even when the solid phase is paramagneticparticles (PMP), assays are typically processed by magneticallycapturing the micro particles and exchanging solutions in a singlecontainer. Embodiments of the invention use multiple chambers to holdthe test sample and water-based or water-alcohol mixture wash buffersand the buffer for carrying out analysis. The water based solutions inthe chambers are separated by a lipophilic material (e.g., a wax or oil)which is immiscible with the solutions. This is illustrated, in someembodiments, with a wax material and wells. The wax in the differentwells connects to each other forming a wax channel (FIG. 1). The wax canbe solidified for storage and transport. During the assay, the wax ismelted and the PMP are dragged up into the wax and moved from onecompartment to the next by passing them through the wax. Magnetic fieldsare used to generate the force required to move the PMP and pass themthrough the interface between the water based solution and the wax. Themaximum force is used to move the particles across the interface and aflexible top plate is used to get the magnet close to the interface andreduce the strength of the magnet required to generate the force.

Moving PMP instead of fluids eliminates the need for pumps andaspirators in automated processors and the need of trained technician toaliquot liquids. The use of wax eliminates the need for valves betweencompartments in single-use test cartridges used in point-of-careanalyzers. It also reduces the amount of inhibitors carried over fromone chamber to the next by reducing the amount of liquid being carriedover from one well to the next. Since the force used to move theparticles is magnetic, the system can be completely closed,significantly reducing the risk of contamination, which is a majorproblem in a sensitive assay such as PCR.

I. Devices

As described above, the present invention provides the ability toproduce and use low cost devices for sample preparation and analysis. Insome embodiments, the devices are single-use or multiple use anddisposable. In some embodiments, the devices utilize a plastic (or othermaterial) cartridge comprising a plurality of sample processing (e.g.,sample preparation and/or analysis) wells. Each well comprises a reagentfor sample preparation or analysis. The nature of the reagents dependson the particular sample and analysis methods to be employed. In someembodiments, the cartridge is composed of any material that ischemically inert and provides adequate mechanical strength. In someembodiments, the cartridge is constructed using a foil laminate thatcomprises an aluminum layer for vapor barrier purposes and inert polymerlayers in contact with reagents. In embodiments that involve thepurification and analysis of RNA (e.g., viral detection or load assays),it is preferred that the cartridge be RNA and RNAse free. Sterilizationmethods known in the art can be utilized to sterilize cartridges priorto use.

The cartridges of embodiments of the present invention are covered witha material that segregates the sample processing chambers. In someembodiments, the material is any lipophilic material that has phasechange characteristics and is immiscible with the reagents for samplepreparation and analysis and substances in the sample which caninterfere with amplification and detection. In some embodiments, thematerial is a wax. In some embodiments, the wax is a liquid at roomtemperature. In other embodiments, the wax is a solid at roomtemperature and a liquid at a temperature suitable for reactions. Inother embodiments, the lipophilic material is an oil. The lipophilicmaterial may be selected as optimal for use with a particular moleculeof interest in terms of temperature use, size exclusion, stability, andthe like.

In some embodiments, the lipophilic material separates the sampleprocessing chambers. In other embodiments, the lipophilic material islocated in between chambers but does not form the physical barrierbetween the chambers. In such embodiments, the sample may pass throughair or other reagents before or after passing through the lipophilicmaterial.

The present invention is not limited to a particular lipophilicmaterial. In some embodiments, liphophilic materials are immiscible inwater and alcohol, exhibit low solubility in water (e.g., ppm), arechemically inert, have melting and boiling points compatible with assayprocessing (for example, perfluorohexame has a by of 56° C.), have aspecific gravity different from water (e.g., float or sink in water),have a low coefficient of expansion, and are stable at 50° C. for longperiods of time (e.g., weeks, months, or years).

Commercially available lipophilic materials that find use in embodimentsof the invention include, but are not limited to, Chill-Out 14 wax (MJResearch), paraffin waxes such as IGI 1070A, microcrystalline waxes suchas IGI Micosere 5788A, soy and palm waxes such as IGI R2322A, candlewaxes such as IGI 6036A, thermoset waxes such as IGI Astorstat 75, hotmelt adhesives, atactic polypropylene and polyolefin compounds,petroleum waxes, and dental waxes.

In other embodiments, natural waxes such as animal waxes (e.g., beeswax,lanolin, or) tallow, vegetable waxes (e.g., carnauba, candelilla, andsoy) or mineral waxes such as fossil or earth (e.g., ceresin or montan)or petroleum (e.g., paraffin or microcrystalline) waxes are utilized. Inyet other embodiments, synthetic (man-made) waxes such as ethylenicpolymers (e.g., polyethylene or polyol ether-esters), chlorinatednaphthalenes or hydrocarbon type waxes (e.g. Fischer-Tropsch) areutilized.

In some embodiments, oils such as mineral oil, paraffin oil, siliconoil, fluorosilicone, fluorocarbon oil (e.g., Fluorinert FC-40 from 3M),perfluorocarbon fluids (e.g., Flutec® Fluids from F2Chemicals),perfluorodecalin (e.g., P9900 from Aldrich, Flutec PP6, FluoroMedAPF-140HP), perfluoroperhydrophenanthrene (e.g., FluoroMed APF-215M) orperfluorooctylbromide (e.g., FluoroMed APF-PFOB) are utilized.

Additional barrier materials include, but are not limited to,1,4-Dioxane, acetonitrile, ethyl acetate, tert-butanol, cyclohexanone,methylene chloride, tert-Amyl alcohol, tert-Butyl methyl ether, butylacetate, hexanol, nitrobenzene, toluene, octanol, octane, propylenecarbonate, and tetramethylene sulfone (See e.g., Chin et al.,Biotechnology and Bioengineering 44:140 (1994); herein incorporated byreference in its entirety).

In still further embodiments, ionic liquids (e.g., BMIM[PF6], BMIM[Tf2N]and OMA[Tf2N] where: BMIM-bis(trifluoromethanesulfonyl)imide,PF6=1-n-butyl-3-methylimidazolium hexafluorophosphate,TfN2=bis(trifluoromethylsulfonyl)imide, and OMA=methyltrioctylammonium)are utilized as barrier materials. In yet other embodiments,1-Butyl-3-methylimidazolium tetrafluoroborate ECOENG™ 21M,1-Ethyl-3-hydroxymethylpyridinium ethylsulfate,Butylmethylpyrrolidiniumbis(trifluoromethylsulfonyl)imide, ECOENG™ 212,or ECOENG™ 1111P (all available from Solvent Innovations) are utilizedas barrier materials.

The reagents provided in the different compartments of the device may beany reagent for performing sample preparation and analysis. Examplesinclude, but are not limited to, cell lysis buffer, wash buffers,affinity reagents, elution buffers, and reaction components forbiological assays. The devices of the present invention are suitable forthe purification of a variety of biological molecules including, but notlimited to, nucleic acids (e.g., RNA, genomic DNA, oligonucleotides andthe like), proteins (e.g., peptides, peptide fragments, oligomericproteins, protein complexes, membrane proteins, and the like), andantibodies. The devices of the present invention are suitable forcarrying out any number of biological assays including, but not limitedto, amplification of RNA or DNA (e.g., PCR, TMA, NASBA), detection ofnucleic acids (e.g., hybridization assays), and immunoassays.

In some embodiments, the device includes a component to transport samplefrom one compartment of the device to the next. In some embodiments,samples are associated with magnetic beads and the transport componentis a magnet. In other embodiments, the transport component generateselectric current to transport sample. In still further embodiments,centrifugal force is utilized to transport sample and the transportcomponent generates such force (e.g., by movement of the device). Inother embodiments, a fluid with a specific gravity greater than water isused such that the fluid moves without mechanical intervention.

In some embodiments, the device includes a detection component to detecta labeled or otherwise presence biological sample or assay product.Examples include, but are not limited to, spectrophotometers, massspectrometers, NMR, microscopy and the like. In some embodiments,products are read directly from the final compartment of the device(e.g., using a window for spectroscopy). In other embodiments, productsare removed from the device (e.g., using an automated component of thedevice) for detection.

Embodiments of the present invention further provide a device comprisingfluidic chamber of reactants (e.g., a vertical column) separated by a“wall” of lipophilic material which prevents the reactants from mixingbut allows microparticles to cross (e.g., magnetic particles transportedby a magnet).

In some embodiments, the device and its use are automated. An automatedsystem comprises a device for sample purification and analysis, atransport component for moving sample through the device, and anyadditional components necessary, sufficient or useful for the automationof the process (e.g. pre-processing reagents and sample transport orpost analysis detection or further analysis components). In someembodiments where magnetic transport is utilized, the transportcomponent comprises a magnet that moves between chambers of the device.In other embodiments, the device moves relative to a stationary magnetor other transport device.

II. Methods

As described above, the present invention provides sample preparationand analysis devices and methods of using the devices.

A. Sample

Any sample suspected of containing the desired material for purificationand/or analysis may be tested according to the disclosed methods. Insome embodiments, the sample is biological sample. Such a sample may becells (e.g. cells suspected of being infected with a virus), tissue(e.g., biopsy samples), blood, urine, semen, or a fraction thereof(e.g., plasma, serum, urine supernatant, urine cell pellet or prostatecells), which may be obtained from a patient or other source ofbiological material, e.g., autopsy sample or forensic material.

Prior to contacting the sample with the device or as a component of thedevice or automated system, the sample may be processed to isolate orenrich the sample for the desired molecules. A variety of techniquesthat use standard laboratory practices may be used for this purpose,such as, e.g., centrifugation, immunocapture, cell lysis, and nucleicacid target capture.

In other embodiments, the methods of embodiments of the presentinvention are utilized to purify and/or analyze intact cells (e.g.,prokaryotic or eukaryotic cells).

B. Purification Methods

In some embodiments, the devices of the present invention are utilizedin sample preparation and purification. Any suitable methods forpurification may be utilized, including but not limited to, targetcapture, washes, precipitations and the like. In some embodiments,sample purification is carried out entirely in the device and does notrequire any additional purification steps. Purification may occur in oneor more reaction chambers. This decreases the complexity of purificationand reduces cost. One of skill in the art recognizes that the particularpurification method is dependent on the nature of the target biologicalsample.

C. Modification/Analysis/Detection

The purified sample may be detected using any suitable methods,including, but not limited to, those disclosed herein. The descriptionbelow provides exemplary techniques for biological molecules such asnucleic acids and proteins. Other techniques may be applied forbiological molecules or non-biological molecules, as desired or needed.

i. Nucleic Acid Detection

Examples of nucleic modification/analysis/detection methods include, butare not limited to, nucleic acid sequencing, nucleic acid hybridization,and nucleic acid amplification. Illustrative non-limiting examples ofnucleic acid sequencing techniques include, but are not limited to,chain terminator (Sanger) sequencing and dye terminator sequencing.Those of ordinary skill in the art will recognize that because RNA isless stable in the cell and more prone to nuclease attack experimentallyRNA is usually reverse transcribed to DNA before sequencing.Illustrative non-limiting examples of nucleic acid hybridizationtechniques include, but are not limited to, in situ hybridization (ISH),microarray, and Southern or Northern blot. Nucleic acids may beamplified prior to or simultaneous with detection.

Illustrative non-limiting examples of nucleic acid amplificationtechniques include, but are not limited to, polymerase chain reaction(PCR), reverse transcription polymerase chain reaction (RT-PCR),transcription-mediated amplification (TMA), ligase chain reaction (LCR),strand displacement amplification (SDA), and nucleic acid sequence basedamplification (NASBA). Those of ordinary skill in the art will recognizethat certain amplification techniques (e.g., PCR) require that RNA bereversed transcribed to DNA prior to amplification (e.g., RT-PCR),whereas other amplification techniques directly amplify RNA (e.g., TMAand NASBA).

The polymerase chain reaction (U.S. Pat. Nos. 4,683,195, 4,683,202,4,800,159 and 4,965,188, each of which is herein incorporated byreference in its entirety), commonly referred to as PCR, uses multiplecycles of denaturation, annealing of primer pairs to opposite strands,and primer extension to exponentially increase copy numbers of a targetnucleic acid sequence. In a variation called RT-PCR, reversetranscriptase (RT) is used to make a complementary DNA (cDNA) from mRNA,and the cDNA is then amplified by PCR to produce multiple copies of DNA.For other various permutations of PCR see, e.g., U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159; Mullis et al., Meth. Enzymol. 155:335 (1987); and, Murakawa et al., DNA 7: 287 (1988), each of which isherein incorporated by reference in its entirety.

Transcription mediated amplification (U.S. Pat. Nos. 5,480,784 and5,399,491, each of which is herein incorporated by reference in itsentirety), commonly referred to as TMA, synthesizes multiple copies of atarget nucleic acid sequence autocatalytically under conditions ofsubstantially constant temperature, ionic strength, and pH in whichmultiple RNA copies of the target sequence autocatalytically generateadditional copies. See, e.g., U.S. Pat. Nos. 5,399,491 and 5,824,518,each of which is herein incorporated by reference in its entirety. In avariation described in U.S. Publ. No. 20060046265 (herein incorporatedby reference in its entirety), TMA optionally incorporates the use ofblocking moieties, terminating moieties, and other modifying moieties toimprove TMA process sensitivity and accuracy.

The ligase chain reaction (Weiss, R., Science 254: 1292 (1991), hereinincorporated by reference in its entirety), commonly referred to as LCR,uses two sets of complementary DNA oligonucleotides that hybridize toadjacent regions of the target nucleic acid. The DNA oligonucleotidesare covalently linked by a DNA ligase in repeated cycles of thermaldenaturation, hybridization and ligation to produce a detectabledouble-stranded ligated oligonucleotide product.

Strand displacement amplification (Walker, G. et al., Proc. Natl. Acad.Sci. USA 89: 392-396 (1992); U.S. Pat. Nos. 5,270,184 and 5,455,166,each of which is herein incorporated by reference in its entirety),commonly referred to as SDA, uses cycles of annealing pairs of primersequences to opposite strands of a target sequence, primer extension inthe presence of a dNTPaS to produce a duplex hemiphosphorothioatedprimer extension product, endonuclease-mediated nicking of ahemimodified restriction endonuclease recognition site, andpolymerase-mediated primer extension from the 3′ end of the nick todisplace an existing strand and produce a strand for the next round ofprimer annealing, nicking and strand displacement, resulting ingeometric amplification of product. Thermophilic SDA (tSDA) usesthermophilic endonucleases and polymerases at higher temperatures inessentially the same method (EP Pat. No. 0 684 315).

Other amplification methods include, for example: nucleic acid sequencebased amplification (U.S. Pat. No. 5,130,238, herein incorporated byreference in its entirety), commonly referred to as NASBA; one that usesan RNA replicase to amplify the probe molecule itself (Lizardi et al.,BioTechnol. 6: 1197 (1988), herein incorporated by reference in itsentirety), commonly referred to as Qβ replicase; a transcription basedamplification method (Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173(1989)); and, self-sustained sequence replication (Guatelli et al.,Proc. Natl. Acad. Sci. USA 87: 1874 (1990), each of which is hereinincorporated by reference in its entirety). For further discussion ofknown amplification methods see Persing, David H., “In Vitro NucleicAcid Amplification Techniques” in Diagnostic Medical Microbiology:Principles and Applications (Persing et al., Eds.), pp. 51-87 (AmericanSociety for Microbiology, Washington, D.C. (1993)).

Non-amplified or amplified target nucleic acids can be detected by anyconventional means. For example, target mRNA can be detected byhybridization with a detectably labeled probe and measurement of theresulting hybrids. Illustrative non-limiting examples of detectionmethods are described below.

One illustrative detection method, the Hybridization Protection Assay(HPA) involves hybridizing a chemiluminescent oligonucleotide probe(e.g., an acridinium ester-labeled (AE) probe) to the target sequence,selectively hydrolyzing the chemiluminescent label present onunhybridized probe, and measuring the chemiluminescence produced fromthe remaining probe in a luminometer. See, e.g., U.S. Pat. No. 5,283,174and Norman C. Nelson et al., Nonisotopic Probing, Blotting, andSequencing, ch. 17 (Larry J. Kricka ed., 2d ed. 1995, each of which isherein incorporated by reference in its entirety).

Another illustrative detection method provides for quantitativeevaluation of the amplification process in real-time. Evaluation of anamplification process in “real-time” involves determining the amount ofamplicon in the reaction mixture either continuously or periodicallyduring the amplification reaction, and using the determined values tocalculate the amount of target sequence initially present in the sample.A variety of methods for determining the amount of initial targetsequence present in a sample based on real-time amplification are wellknown in the art. These include methods disclosed in U.S. Pat. Nos.6,303,305 and 6,541,205, each of which is herein incorporated byreference in its entirety. Another method for determining the quantityof target sequence initially present in a sample, but which is not basedon a real-time amplification, is disclosed in U.S. Pat. No. 5,710,029,herein incorporated by reference in its entirety.

Amplification products may be detected in real-time through the use ofvarious self-hybridizing probes, most of which have a stem-loopstructure. Such self-hybridizing probes are labeled so that they emitdifferently detectable signals, depending on whether the probes are in aself-hybridized state or an altered state through hybridization to atarget sequence. By way of non-limiting example, “molecular torches” area type of self-hybridizing probe that includes distinct regions ofself-complementarity (referred to as “the target binding domain” and“the target closing domain”) which are connected by a joining region(e.g., non-nucleotide linker) and which hybridize to each other underpredetermined hybridization assay conditions. In a preferred embodiment,molecular torches contain single-stranded base regions in the targetbinding domain that are from 1 to about 20 bases in length and areaccessible for hybridization to a target sequence present in anamplification reaction under strand displacement conditions. Understrand displacement conditions, hybridization of the two complementaryregions, which may be fully or partially complementary, of the moleculartorch is favored, except in the presence of the target sequence, whichwill bind to the single-stranded region present in the target bindingdomain and displace all or a portion of the target closing domain. Thetarget binding domain and the target closing domain of a molecular torchinclude a detectable label or a pair of interacting labels (e.g.,luminescent/quencher) positioned so that a different signal is producedwhen the molecular torch is self-hybridized than when the moleculartorch is hybridized to the target sequence, thereby permitting detectionof probe:target duplexes in a test sample in the presence ofunhybridized molecular torches. Molecular torches and many types ofinteracting label pairs are known (e.g., U.S. Pat. No. 6,534,274, hereinincorporated by reference in its entirety).

Another example of a detection probe having self-complementarity is a“molecular beacon” (see U.S. Pat. Nos. 5,925,517 and 6,150,097, hereinincorporated by reference in entirety). Molecular beacons includenucleic acid molecules having a target complementary sequence, anaffinity pair (or nucleic acid arms) holding the probe in a closedconformation in the absence of a target sequence present in anamplification reaction, and a label pair that interacts when the probeis in a closed conformation. Hybridization of the target sequence andthe target complementary sequence separates the members of the affinitypair, thereby shifting the probe to an open conformation. The shift tothe open conformation is detectable due to reduced interaction of thelabel pair, which may be, for example, a fluorophore and a quencher(e.g., DABCYL and EDANS).

Other self-hybridizing probes are well known to those of ordinary skillin the art. By way of non-limiting example, probe binding pairs havinginteracting labels (e.g., see U.S. Pat. No. 5,928,862, hereinincorporated by reference in its entirety) may be adapted for use in thecompositions and methods disclosed herein. Probe systems used to detectsingle nucleotide polymorphisms (SNPs) might also be used. Additionaldetection systems include “molecular switches,” (e.g., see U.S. Publ.No. 20050042638, herein incorporated by reference in its entirety).Other probes, such as those comprising intercalating dyes and/orfluorochromes, are also useful for detection of amplification productsin the methods disclosed herein (e.g., see U.S. Pat. No. 5,814,447,herein incorporated by reference in its entirety).

In some embodiments, detection methods are qualitative (e.g., presenceor absence of a particular nucleic acid). In other embodiments, they arequantitative (e.g., viral load).

ii. Protein Detection

Examples of protein detection methods include, but are not limited to,enzyme assays, direct visualization, and immunoassays. In someembodiments, immunoassays utilize antibodies to a purified protein. Suchantibodies may be polyclonal or monoclonal, chimeric, humanized, singlechain or Fab fragments, which may be labeled or unlabeled, all of whichmay be produced by using well known procedures and standard laboratorypractices. See, e.g., Burns, ed., Immunochemical Protocols, 3^(rd) ed.,Humana Press (2005); Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory (1988); Kozbor et al., Immunology Today 4:72 (1983); Köhler and Milstein, Nature 256: 495 (1975). In someembodiments, commercially available antibodies are utilized.

D. Data Analysis

In some embodiments, following purification and detection, acomputer-based analysis program is used to translate the raw datagenerated by the detection assay (e.g., the presence, absence, or amountof a given target molecule) into data of predictive value for aclinician or researcher. In some embodiments, the software program isintegrated into an automated device. In other embodiments, it isremotely located. The clinician can access the data using any suitablemeans. Thus, in some preferred embodiments, the present inventionprovides the further benefit that the clinician, who is not likely to betrained in genetics or molecular biology, need not understand the rawdata. The data is presented directly to the clinician in its most usefulform. The clinician is then able to immediately utilize the informationin order to optimize the care of the subject.

Any method may be used that is capable of receiving, processing, andtransmitting the information to and from laboratories conducting theassays, information provides, medical personal, and subjects. Forexample, in some embodiments of the present invention, a sample (e.g., abiopsy or a serum or urine sample) is obtained from a subject andsubmitted to a service (e.g., clinical lab at a medical facility,genomic profiling business, etc.), located in any part of the world(e.g., in a country different than the country where the subject residesor where the information is ultimately used) to generate raw data. Wherethe sample comprises a tissue or other biological sample, the subjectmay visit a medical center to have the sample obtained and sent to theprofiling center, or subjects may collect the sample themselves (e.g., aurine sample) and directly send it to a profiling center. Where thesample comprises previously determined biological information, theinformation may be directly sent to the profiling service by the subject(e.g., an information card containing the information may be scanned bya computer and the data transmitted to a computer of the profilingcenter using an electronic communication systems). Once received by theprofiling service, the sample is processed and a profile is produced(i.e., expression data), specific for the diagnostic or prognosticinformation desired for the subject.

The profile data is then prepared in a format suitable forinterpretation by a treating clinician. For example, rather thanproviding raw data, the prepared format may represent a diagnosis orrisk assessment (e.g., viral load levels) for the subject, along withrecommendations for particular treatment options. The data may bedisplayed to the clinician by any suitable method. For example, in someembodiments, the profiling service generates a report that can beprinted for the clinician (e.g., at the point of care) or displayed tothe clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point ofcare or at a regional facility. The raw data is then sent to a centralprocessing facility for further analysis and/or to convert the raw datato information useful for a clinician or patient. The central processingfacility provides the advantage of privacy (all data is stored in acentral facility with uniform security protocols), speed, and uniformityof data analysis. The central processing facility can then control thefate of the data following treatment of the subject. For example, usingan electronic communication system, the central facility can providedata to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the datausing the electronic communication system. The subject may chose furtherintervention or counseling based on the results. In some embodiments,the data is used for research use. For example, the data may be used tofurther optimize the inclusion or elimination of markers as usefulindicators of a particular condition or stage of disease.

E. Compositions & Kits

In some embodiments, systems and/or devices of the present invention areshipped containing all components necessary to perform purification andanalysis (e.g., pre-loaded into the device). In other embodiments,additional reaction components are supplied in separate vessels packagedtogether into a kit.

Any of these compositions, alone or in combination with othercompositions disclosed herein or well known in the art, may be providedin the form of a kit. Kits may further comprise appropriate controlsand/or detection reagents. Any one or more reagents that find use in anyof the methods described herein may be provided in the kit.

EXPERIMENTAL

The following examples are provided to demonstrate and illustratecertain preferred embodiments and aspects of the compositions andmethods disclosed herein, but are not to be construed as limiting thescope of the claimed invention.

Example 1

This example described moving paramagnetic micro particles usingmagnetic forces to capture RNA, subsequent purification, and release ofRNA for amplification and detection. It also shows the use of liquidwax, as an exemplary lipophilic material, as a valve between the variouschambers allowing for the movement of paramagnetic micro particles whileforming a barrier between the various wash buffer.

(a) Fabrication of the Cartridge

Individual cartridges for the assay were prepared by machining asterile, clear round bottom polystyrene 96 well plate (FIG. 1 a). Thewax channel was made using FDA compliant plain back 1/16″ polypropylenesheets. In order to fabricate the wax channel, 9495 LE acrylic adhesivetransfer tape (3M, St. Paul, Minn.) meant for low surface energysubstrates was glued onto both sides of a rectangular piece of plasticcut to the correct dimensions. A rectangular channel was milled out ofthe plastic piece. The milling process also cut the adhesive. In view ofthe poor thermal conductivity and low melting point of polypropylene,special care has to be taken while machining it. The burrs formingduring machining of the channel were removed using a razor blade. Thetop plate is also made out of a polypropylene sheet. Tiny holes werepunched on the top plate to allow for the introduction of fluids.

Polypropylene was used for the top plate and the channel because of itsexcellent chemical resistance. The saturated olefinic chains yieldresistance to most oils and solvents, as well as water-based chemicals,soaps, and moderate acids and bases. Few other materials with thestrength properties of polypropylene match the chemical resistance ofpolypropylene. Also, polypropylene's hard, high-gloss surface makes itdesirable for environments where there is concern for build-up that caninterfere with flow sterilization of the cartridge: it is preferred thata RNA free environment is maintained throughout the test. Whilecommercially available plastic parts are radiated with gamma rays tosterilize them, a protocol was developed to sterilize the plastic partswhich could be easily carried out in a laboratory setting. The wells tobe used for the test were sealed off using adhesive tape beforemachining the plate. After the completion of machining, the cartridgewas washed using the following protocol:

a. wash twice with water

b. wash twice with 100% ethyl alcohol

c. wash with RNaseZap® RNase Decontamination Solution (Ambion, Austin,Tex.)

d. wash twice with water

After the washing was complete, the cartridge was dried overnight at 50°C.

RNA Purification Assay:

The assay was performed using 400 μL of plasma sample containing 10⁶copies/mL Armored RNA (Abbott Molecular, Des Plaines, Ill.). Armored RNAis used instead of naked RNA as a test sample because it is ribonucleaseresistant and easily quantifiable by copy number of RNA. It is alsononinfectious, making it easy for to handle.

In order to purify RNA from a plasma sample, the MagMAX Viral RNAIsolation Kit (Ambion, Austin, Tex.) was employed. In this method, thecells are disrupted using the classic method of guanidiumthiocyanate-based solution. This simultaneously releases the viral RNAand deactivates the nucleases in the sample matrix. The RNA then bindsto the silica coated magnetic beads in the presence of a chaotropicagent and alcohol. The beads are then washed and eluted in aqueous lowsalt buffer.

Preparation of lysis/binding solution and bead mix: Carrier RNA is addedto the lysis/binding solution concentrate according to Table 1 and mixedbriefly. This is followed by the addition of 100% isopropanol. In orderto prepare the bead mix, 10 μL of RNA binding beads are mixed with 10 μLof lysis enhancer for every reaction. The beads are vortexed beforealiquoting.

TABLE 1 Volume of reagents to be added to prepare lysis buffer ReagentAmount Lysis/Binding soln. Concentrate 400 μL Carrier RNA  2 μL 100%isopropanol 400 μL

Preparation of the Cartridge

802 μL of the lysis solution was added to a microfuge tube along with400 μL of plasma sample containing armored RNA. When adding sample, thepipette tips should be immerged slightly into the solution to preventaerosol formation leading to contamination. The solution is vortex mixedgently for 30 seconds and then 20 μL of bead mix is added to the tube.The solution is vortex mixed gently for 4 minutes on a vortex mixture tofully lyse the viruses and bind RNA to the magnetic beads. The beads arecaptured by leaving the microfuge tube on a magnetic stand. 600 μL, ofthe solution is removed and discarded away. The beads are vortex mixedin the remaining 222 μL, of solution. This solution is aliquoted intothe first chamber of the cartridge. 150 μL of wash buffer 1 containingisopropanol is added to chamber two and three respectively. 225 μL ofwash buffer 2 containing ethanol is added to chamber four and fiverespectively. 50 μL of elution buffer is added to chamber 6.

The wax channel (FIG. 1-b) is now glued onto the cartridge by peelingoff the paper laminate of the adhesive transfer tape attached to the waxchannel and applying the wax channel to the cartridge. This is followedby the adhesion of the top plate to the wax channel following a similarprocess. Chill-Out Liquid Wax (Bio-Rad Laboratories, Hercules, Calif.)is then pipetted into the cartridge through the punched holes of the topplate till there is no air gap remaining in the cartridge. FIG. 1 showsthe image of the completely filled cartridge with the different buffersand the wax.

Sample Purification Protocol

(a) Purification in the cartridge. In the sample purification process, amagnetic force is used to carry out the various purification steps (FIG.2), for example, magnetic separation of the beads to accumulate theminto a clump, movement of the particles from the buffer to the wax,movement of the particles in the wax, reintroduction of the particlesinto the next buffer and agitation of the particles in the buffer(Sample C1 in table 2). This magnetic force is produced by a permanentmagnet. The clump of particles was moved from lysis buffer in chamber 1to elution buffer in chamber 6 through the various wash buffers inchambers 2-5. The particles were allowed to sit for 30 seconds in eachwash buffer, and for 10 minutes in the elution buffer. The particleswere moved magnetically in the buffers during these times.

(b) Purification in a microfuge tube. The sample purification wascarried out using the Ambion MagMax kit in a microfuge tube as well. Inthis case, the microfuge tube containing the lysis buffer and the RNAbinding PMPs was put on a magnetic stand to capture the PMPs. Oncecapture in complete, the RNA binding beads formed a pellet against themagnet in the magnetic stand. The supernatant is aspirated out withoutdisturbing the beads. The tube was removed from the magnetic stand, andWash buffer 1 was added to it. The solution was agitated for 30 seconds.A similar process of capture and aspiration was carried out. The beadswere washed twice with Wash Buffer 1 and twice with Wash Buffer 2. Inthis study, two different combinations of wash buffer volumes were used:(i) 300 μL, of wash buffer 1 and 450 μL of wash buffer 2 (sample C2 intable 2) (ii) 150 μL of wash buffer 1 and 225 μL of wash buffer 2(sample C3 in table 2) After the 4 wash steps the beads were left openat room temperature to allow the remaining alcohol to evaporate. Thetubes were inspected for any remaining alcohol since alcohol inhibitsPCR. 50 μL of elution buffer was added and the sample agitatedvigorously for 4 minutes. In each case, the particles are captured afterelution and 12.5 μL of the sample was used for RT-PCR using the AbbottReal time Assay.

Results and Data Analysis

The table shown below shows the average Ct values for the three Sampletypes

TABLE 2 Samples and the Ct values for the Sample Average Ct valuePrototype cartridge + reduced wash buffer 15.2 (C1) Ambion kit protocolin microfuge tube 16.7 Ambion kit in microfuge tube + reduced 20.1 washbuffer (C3)

The data shows that there is a 2.8 fold (2̂mean Ct difference)improvement in RNA purification when the particles are moved throughwax. This occurs in spite of the reduction in the wash buffer volume.Comparing the results with a similar volume of wash buffer, there is a30 fold improvement in RNA purification. In the manual approach ofsample purification, a significant amount of solution would adhere tothe particles, which cannot be aspirated out. Therefore, while each washstep leads to the dilution of the inhibitors which are carried from thelysis solution, it would take a significant number of washes and washbuffer volume to dilute out all the inhibitors completely. While usingwax as a medium of particles transport, the amount of liquid beingcarried is significantly lower which leads to an improvement in Ct.

Effect of Alcohol on RT-PCR

Alcohol is a known inhibitor of PCR. Therefore, the Ambion samplepurification kit requires drying the particles in air before eluding theRNA. In the new protocol described herein, the drying step is completelyremoved. Again, this shows that the amount of fluid being carried fromone chamber to the next is minimal. It also simplifies the samplepurification process significantly allowing for easy automation. Thepresence of smaller quantities of inhibitors also allows for elution insmaller quantities of elution buffer, which in turn speeds up thethermal cycling speed, thereby reducing the time required to carry outthe test.

Example 2 Automation

An inexpensive automated sample purification system is developed bymoving magnetic particles instead of fluids. A cartridge which can holdfluid through long periods of storage is designed. This does away withany sort of pipetting in the field. Optimization of the assay in thisnovel platform enables one to improve the sample purification processand develop a better understanding of the system. An automated systemfor carrying out the purification allows for the measurement of viralload without the need of highly trained lab technicians. The combinationof a closed cartridge and automated device would create a point of careplatform not only for HIV viral load, but for all kinds of nucleic acidtesting in a cheap, convenient manner with a reduced risk of crosscontamination.

Experiments to Optimize the System Include:

a Fabricate a cartridge using foil laminate to hold the chemicals forstorage and to carry out reactions.

b Optimization of the sample purification protocol in new cartridge.

c Build a robust automated system to remove man-power requirements

a. Fabricate a Prototype Cartridge to Carry out Sample Purification

There are two parts to this fabrication: (a) Material used to make thecartridge (b) design of the cartridge itself

(a) Material. The material to be used to make the cartridge preferablyprovides (a) a vapor barrier to hold the liquids through long periods ofstorage without significant loss. (b) mechanical strength; and (c) achemically inert surface which does not stick to the blood or particlespresent in the solution.

While polystyrene or polypropylene is the choice of material forcarrying out chemical assays because of their chemical inertness, theydo not provide the vapor barrier needed to store the reagents for longperiods of time. The vapor barrier of a material is measured in terms ofwater vapor transmission rate (WVTR) which is a measure of the passageof water vapor through a substance.

WVTR=Δw/ΔtA(gm ⁻² s ⁻¹),

Where Δw/Δt is the amount of moisture loss per unit time of transfer (gs⁻¹) and A is the exposed area to moisture transfer (m²)

TABLE 3 WVTR characteristics of some common plastics Material Density(g/cm3) Thickness (mil) WVTR^(a) WVTR^(b) Ultra Low 0.9015 10 0.33130.846 density poly Ethylene Low density poly 0.9188 2 0.17 0.4274ethylene High density 0.9433 2.5 0.053 0.136 poly ethylene Polypropylene 0.8994 2.5 0.1015 0.2738 Rollprint foil 2.1011 3.5 0 0laminate ^(a)g/mil/100 sq in/day at 30° C. and 35% RH ^(b)g/mil/100 sqin/day at 40° C. and 35% RH

Table 3 shows table below shows the WVTR of some plastics and of a foillaminate (RP#26-1244, Rollprint, Addison, Ill.). It is evident that foillaminate provides excellent vapor barrier for reagents during storage.Foil laminate has the added benefit of being extremely inexpensive. Thecost of a single cartridge made out of foil laminate would not exceed afew cents. The various layers of foil laminate to be used are shown inFIG. 3 below.

The foil laminate (Rollprint Packaging Products, Addison, Ill.) containsa 2 mil Aluminum layer which is responsible for the low WVTR making itideal for the storage of reagents. The polyester layer provides achemically inert and hydrophobic surface preferred for carrying out theassay. It also allows the foil to be heat sealed to another piece offoil or plastic. The nylon outer surface protects the aluminum surfacefrom corrosion and also provides mechanical strength to the foillaminate. The foil laminate lacks rigidity. This can be overcome bypackaging it in a rigid material. It should be noted that while the foillaminate is virtually impermeable to vapor, the seal between two layersof foil is not and is responsible for some loss of vapor.

In other embodiments, a vacuum formed chamber made out of polypropyleneor polystyrene similar to a 96 well plate is used. In some embodiments,in order to provide a vapor barrier, this would be aluminized by vapordeposition.

(b) Shape and size of the cartridge. While the shape of the chambers aregoverned by the various forces acting to move the particles and theneeds to simplify the automation, the size of the chambers is governedby the chemistry associated with sample purification. The prototypedesigning is done using a 3D Mechanical CAD program, Solidworks(Solidworks Corporation, Concerd, Mass.). An example of a cartridgebased on the currently used volumes is shown in FIG. 4. Changes in thechemistry or automation needs are accommodated by changing the shape andsize of the chambers accordingly.

(c) Peelable layer and top plate: The top plate of the cartridge ispreferably transparent in order to make optical readings during thethermal cycling. Not shown in the drawing is a peelable foil laminatelayer which sits between the top plate and the foil chambers. This issimilar to a printer ink cartridge. The end of the peel comes outbetween the top plate and the wax layer. This provides the vapor barrieroptimum for long term storage. Solid wax is present above and below theseal. The wax below allows for the peel to be removed without risk ofloss of reagent sticking to the peel. Before the test, the peel isremoved and the wax melted. The top plate can be made flexible whichallows us to compress it to remove any air gap that forms because of thecavity created by the removal of the seal. It also should be chemicallyinert since it comes in contact with the particles when they are draggedfrom one chamber to the next. The peelable seal is heat sealed to thechambers. The distance between the chambers is preferably 6 mm to allowfor proper sealing, although other dimensions may be used. This sealingprocess is carried out in Rollprint Inc.

Fabrication. The fabrication of the aluminum foil cartridges is doneusing a pinch press device that comprises a positive hemispherical headwhich fits into a negative mold of the same size. By pressing the foilbetween the positive head and the negative mold, the foil is stretchedto confirm with the shape of the mold. While the yield strength and thetensile strength of the laminate are not known, the standard industrypractice is:

(Maximum area after making the chamber)<(2×area before extension)

The wax channel, the solid support, for cartridge, the top plate is alljoined together using 3M adhesive transfer tape. Several adhesives weretested before choosing this one because of its excellent adhesionproperties, especially to low surface energy surfaces. The adhesive islaser cut to the correct size by GML (Vadnais Heights, Minn.).Optimization of sample purification protocol to improve assayperformance:

The assay is performed using a silica coated PMP based MagMax kit fromAmbion. The kit protocol is optimized for best performance in an assayformat which involves sedimentation of the beads and pipetting out ofthe fluid. It is not meant to be used with the wax. In order to furtherunderstand the effects of the wax on the sample purification and tooptimize the sample purification process, the following experiments arecarried out:

1. Carryover of water through the wax: Based on the initial experiments,it was hypothesized that the carry-over of water or buffer from onechamber to the next is reduced significantly on moving the particlesthrough the wax. This was based, in part, on the results describedabove, which show the lack of inhibition due to alcohol. In order toconfirm this hypothesis, a known concentration of a fluorescent dye,fluorescein is dissolved in water. A known number of particles (asmeasured on a Luminex flow cytometer) is added to this solution. Then,the particles are magnetically moved through the wax into a known volumeof fluorescein free water. On measuring the concentration of fluoresceinin the starting sample and the final sample, one can quantitativelyestimate the volume of water being carried over from one chamber to thenext (V_(a)).

The experiment has two controls which are used to compare the results:(a) move the particles with a pick-pin from an identical solution offluorescein to water. This represents currently available systems suchas the Maxwell or Kingfisher. (b) Collect the particles by putting themicrofuge tube containing the fluorescein solution and the particles ona magnetic stand and then carefully pipetting out the solution. This isfollowed by the addition of a fixed quantity of fluorescein free water.

An improvement in purification performance is quantified by comparingthe average volume of liquid carried across from one chamber to thenext. The variance is used to quantify the variability in thepurification process due to sample handling. The experiment is repeatedfor different number of particles, different initial volume of sample,and different fluorescein concentrations. The volume of liquid beingcarried across is a fraction Φ of the volume of the droplet (V)containing the particles which moved into the wax.

(1−Φ)V=(4/3)πr ³ N

ΦV=V _(a)

Φ=V _(a)/(Va+4πr ³ N)

In the above equations, r is the radius of the particles and N is thetotal number of particles. The volume of the droplet V is dependant on Nand therefore the volume of liquid ΦV is also dependant of N. One wouldnot expect ΦV to be dependent on the initial volume of fluoresceinsolution, but rather on the concentration of fluorescein. Thisexperiment allows one to determine the optimal wash steps and volume, orthe volume of the lysis buffer required.

2. Selecting optimal wash step volume and number. The protocolrecommended by Ambion is optimized for a manual pipetting procedure. Itdoes not account for the presence of the wax. Therefore, the protocol isoptimized using wax. While all the inhibitors present in blood plasmawhich inhibit PCR are not known specifically, it is accepted thatinhibition by most inhibitors is concentration dependant. The resultsfrom the previous experiments provide information about the effect ofthe wax on the removal of fluids. Using that as a guide stick, thenumber of purification steps is reduced. The reduction in the number ofpurification steps reduces the complexity of the sample purificationprocess, reduces the cost of the test and the time required for thetest. A reduction in the inhibitor concentration allows for elution in asmaller volume of elution buffer, leading to a further increase in theRNA concentration and faster thermal cycling.

The sensitivity of a method is associated with the lower limit ofapplicability of that method. In relation to chemicals, the minimumdetectable value often refers to the minimum detectable netconcentration or amount.

Determination of the LOD

Calculation of the LOD differs between professional scientific bodiesand between different applications, but one definition of the LOD is:

LOD·(mean of blanks)+K(sd)

Where mean of blanks=the mean value given to the blank determinationsassociated with an assay; K=coverage factor associated with a desiredconfidence level; and sd=standard deviation of the blank determinations.

This calculation is unsuitable for estimating the LOD associated withreal-time quantitative PCR methods as the blank controls typically havevalues equal to the highest cycle number used in the PCR reaction.Additionally, if the assay has worked correctly, the value of theseblanks will all be the same and their distribution truncated, thusprecluding the calculation of a useful standard deviation.

In order to overcome some of the difficulties encountered with thetraditional calculation of LOD described in the scientific literature,one can define the LOD as the lowest copy number that gives a detectablePCR amplification product at least 95% of the time. This can also beinterpreted as the lowest copy number that can be distinguished from thebackground noise with a probability of 95%. The current Rt-PCR assayusing the Ambion kit for RNA purification and Abbott Real time protocolfor Rt-PCR has been reported to have an LOD of 40 copies of RNA in 1.0ml of plasma sample. Preferred assays detect a minimum of 500 copies ofRNA. Thus, for every protocol, the sensitivity of the assay is measuredby creating a dilution curve of Ct verses copies of RNA. The desiredgoal is to go from the lysis buffer to the elution buffer without asingle wash step. The purification assay is carried out with differentnumber of wash steps and wash volume.

3. Wax Melting Point

The wax that used in the experiments described in Example 1 has amelting point of 10 C. Therefore, this wax is liquid at roomtemperature. A wax (DyNAwax Reagent, Finland), which melts at 60 C orother higher melting temperature was is used in some embodiments. Thisallows for storage of the wax as solid during storage with subsequentmelting just before the experiment, thereby creating a phase change plugfor the movement of particles. The other advantage is that the presenceof wax below the peelable seal allows one to peel the foil withoutlosing any fluid from the chambers which might have stuck to the foil.However, this would involve carrying out the experiment in a water bathor using a peltier heater to heat the wax.

Automation of the Sample Purification Protocol:

An automated sample purification system is provided. Automation hasnumerous benefits, namely:(a) the process does not require a skilledworker, (b) provides for better understanding of the system, (c) speedsup the assay development and testing process, (d) reduces sample tosample variations by standardizing the process.

(a) A first component is a stage to move magnets and cartridge. In orderto automate the process, a stage is built to carry out the fiveprocesses, namely (a) Aggregation of the particles in a fluid (b)dragging the fluid across the interface (c) dragging the particleaggregate in the wax (d) dragging the particle aggregate from the wax tothe water (e) agitating the particles in the fluid. In the manual modeof operation, the cartridge was held steady while the magnet wasdisplaced relative to the cartridge. In order to create the complexmotion automatically, in some embodiments, the cartridge is moved withrespect to the magnets. Thus the magnets and/or the cartridge aremounted on a moving stage. The decision on which to move is dependent onease of construction, cost and reliability of the process. Steppermotors may be used to carry out the various movements.

i) Aggregate the particles in the wash buffer. The design is guided by asimple estimation of the forces involved. The motion of a sphericalmagnetic particle of density P_(p), radius R_(p), volumeV_(p)=(4πRp³)/3, and mass m_(p) is governed by several forces including,(a) the magnetic force due to all field sources, (b) fluidic drag, (c)particle/fluid interactions (perturbations to the flow field), (d)buoyancy, (e) gravity, (g) thermal kinetics (Brownian motion), and (h)inter-particle effects such as magnetic dipole interactions. In order toguide the design parameters, the behavior of magnetic particles in lowconcentration and slow flow regimes where the magnetic and viscous dragforces dominate is modeled. Therefore, particle/fluid interactions andinterparticle effects are ignored. The gravitational force, which whileof second order might not be negligible depending on the particle size,is included. According to classical al Newtonian dynamics:

m _(p) =dv _(p) /dt=F _(m) +F _(f) +F _(g),

where, v_(p) is the velocity of the particle, and F_(m), F_(f) and F_(g)are the magnetic, fluidic, interfacial and gravitational forces,respectively. The magnetic force is obtained using an “effective” dipolemoment approach where the magnetized particle is replaced by an“equivalent” point dipole with a moment m_(p.eff) (Furlani and Ng,2006). The force on the dipole (and hence on the particle) is given isgiven by:

F _(m)=μ_(f)(m _(p.eff)·∇)H _(a),

where μ_(f) is the permeability of the transport fluid, m_(p.eff) is the“effective” dipole moment of the particle, and H_(a) is the (externally)applied magnetic field intensity at the center of the particle, wherethe equivalent point dipole is located. If the particle is infree-space, m_(p.eff)=V_(p)M_(p) and the above equation reduces to theusual form F_(m)=μ₀(m_(p)·∇)H_(a), where V_(p) and M_(p) are the volumeand magnetization of the particle, and μ₀=4π×10⁻⁷ is the permeability offree space. FIG. 5 shows the arrangement of a magnet above the fluidicchamber and the surface plot of force on a particle in the x directionand the y direction at a fixed distance from the magnet. The forcecalculation requires a choice of particle size and material propertiesof, Fe₃O₄ (magnetite), which make up the particles. In the calculationshown, it was assumed that Fe₃O₄ has a density p=5000 kg/m³, asaturation magnetization of M=4.78×10⁵ A/m and the particle size is 0.5micrometer. A program was written in Matlab to estimate the magneticforce for a system consisting of one magnet or an array of magnets. Thisenables one to calculate the magnetic force for different magnets andarrangements of magnets. While the current program can only estimateforces due to a linear array of magnets, it is possible to generate aprogram to estimate a more complex arrangement of magnets.

The fluidic force is predicted using the Stokes' law for the drag on asphere in uniform flow, F_(f)=−6πηR_(p)(v_(p)−v_(f)), where η and v_(f)are the viscosity and the velocity of the fluid, respectively. Thegravitational force is given by F_(g)=−V_(p)(ρ_(p)−ρ_(f))gý, where ρ_(p)and ρ_(f) are the densities of the particle and fluid, respectively, andg=9.8 m/s² is the acceleration due to gravity. The gravitation forceacts in the −y direction. The gravitational force is often ignored whenanalyzing the magnetophoretic motion of submicron particles, as it isusually much weaker than the magnetic force.

Plugging the various forces into Newton's equation of motion, it ispossible to predict the approximate time it would require for theparticles to aggregate and even plot the particle trajectories. Thesecalculations are used to guide the design parameters.

(ii) Measure the surface tension and drag the particles across theinterface. The strength of the magnet used in the automated process isgoverned by the strength of the magnetic field required to overcome theinterfacial surface tension. While it is difficult to move one particleacross the interface, a particle clump can be moved across theinterface.

The interfacial force can be estimated as y2πR_(p) where y is theinterfacial tension between wax and buffer. The interfacial force ismeasured using the weight drop method which follows from Tate's law.

This is an accurate method of determining surface tension and perhapsthe most convenient laboratory one for measuring surface tension of aliquid-air or liquid-liquid interface. As illustrated in FIG. 6, theprocedure is to form drops of the wax at the end of a tube, allowingthem to fall into a container containing buffer. The weight of the dropis then used to determine the surface tension. Tate's law gives a verysimple expression for W, the weight of a drop minus the displaced fluid:W=2πryf, where is the radius of the tube from which the drop forms and fis a function of the dimensionless ratio of r/N^(1/3), where V is thedrop volume. The system shown in FIG. 6 is used to measure the surfacetension between the wax and the various wash buffers. An importantprecaution to be taken when employing this method is to use a tip thathas been ground smooth at the end and which is free from any nicks. Inthe case of liquids that do not wet the tip, r is the inner radius. Thedrops should also be formed slowly otherwise the drop weight will behigh.

Having estimated the interfacial force, one can estimate the magneticforce required to move the particles across the interface. Therefore,the design criteria is: F_(m)>F_(i), where F_(m) and F_(i) are themagnetic and interfacial forces respectively. This estimation neglectsfrictional force as well. This estimation guided the choice of magnet.In order to reduce the strength of the magnet required to move theparticles from the buffer to the wax, the magnet is moved as close tothe interface as possible. This is done with a flexible top plate. Aflexible top plate is also used so that it can be depressed to removeany air gaps which may form when the foil laminate which lies betweenthe chambers and the top plate is peeled off.

iii) Drag the particle aggregate across the wax. The equation of motionfor dragging the particles through the wax is similar to that ofaggregation of the particles. However, in this case, the motion is thatof a droplet of fluid containing a particle aggregate rather than thatof a single particle. The net frictional force is given by

F _(f)=−6πηRp(v _(p) −v _(f)),

where R_(p) is the translational tensor which depends on the shape ofthe droplet and the internal viscosity of the droplet. It is fairly easyto drag the particle aggregate through the wax. During this process, theparticles come in contact with the top surface of the cartridge. It istherefore preferred that the material used for the top plate process ischemically inert and has a smooth surface texture.

(iv) Drag the particle aggregate from the wax to the buffer. Since themovement of particles from the wax to the wash buffer is accompanied bya decrease in surface energy, this is a simple process. A small magneticforce is sufficient to drag the particles back into the water. Also, thegravimetric force aids the process of the particle settlement.

(v) Verify the need for agitation: In the assay performed in Example 1,the particles were agitated by moving a magnet around the chamber. Thiswas done since the sample purification process involving manualpipetting required agitation of the particles in the wash buffer byvortex mixing. While creating a variable magnetic field is possible bymoving magnets on a stage, it adds to the cost of the assay. Thus, insome embodiments, agitation is not used.

Software to control the movement of the stepper motors and thecartridge. The USB port of a computer can be connected to a steppermotor controller via a USB to RS232 converter. This allows one to sendcommands to the motor using the hyper terminal. Any serial communicationsoftware such as Docklight can be used to effectively communicate withthe stepper motor using RS232 command sets.

Test the performance of the automated sample purification system. Inorder to test the performance of the sample purification system, theloss of particles is measured. This is compared to a loss of performancein the manual form of the assay and the assay with pipetting of fluids.For this purpose, the particle concentration is measured using theLuminex flow cytometer. Unlike ordinary flow cytometers, the Luminexsystem has a positive flow control system which allows one to countparticles as well as measure the volume of solution used, therebyenabling one to measure the concentration of the bead solution. Loss ofbeads=Initial concentration of bead×Vol of sample used−finalconcentration of beads×Vol of elution buffer.

%loss of beads=(Loss of beads/Total initial number of beads)×100

The percentage loss of beads is measured at each stage of the automationprocess to measure the performance of the system. The aim is to reducethe percentage loss to less than what is observed in a manual process.The mean % loss of beads and the variance of % loss of beads arecompared for the automated process and the manual approach. An automatedsystem is expected to have a lower variance than a manual process.

Example 3 Tubular Processor

This example describes a tubular processor for performing biologicalreactions. The experimental setup for the diagnostic assay is shown inFIG. 7. It consists of a 0.060″ internal diameter tube (Small PartsInc.) attached to a CAVRO 3000XL digital pump. The digital pump iscontrolled through an RS232 interface. The different wash buffers,analytes, solution containing magnetic particles and silicone oil(Gelest Inc.) are pumped in from the distal end of the tube.

The particles used for the experiment are carboxyl coated smooth surfacemagnetic particles obtained from Spherotech Inc. The SPHERO^(1M) SmoothSurface Magnetic Particles have a thick layer of polymer coating on thesurface of the particles to fully encapsulate the iron oxide coating.There is no exposed iron oxide on the surface of the particles.

Cylindrical Neodymium magnets (Bunting Magnetics Co) are moved along thelength of the capillary. The magnets are located around the capillary tomix the particles. Magnets of grade N35 and N40 were used for allexperiments.

Teflon tubes were found to be better than glass tubes and were used forall experiments. The particles stick to the glass more than Teflon. Itis hypothesized that

Teflon, being more hydrophobic than glass, repels the hydrophilicparticles more than glass which is hydrophilic because of the presenceof the carboxyl group on the particle surface. However, the particles donot stick to Teflon because it is extremely hydrophobic.

Fluorescent reading is not taken in the tube for the followingexperiments, although it is possible to attach an optical system to thecapillary system. After completion of the chemical reaction, theparticles are taken out of the tube and read in a flow cytometer. Allreadings are standardized with respect to the SPHERO Rainbow CalibrationParticles. The SPHERO Rainbow Calibration Particles contain a mixture ofseveral similar size particles with different fluorescence intensities.Every particle contains a mixture of fluorophores that allows excitationat any wavelength from 365 to 650 nm. This enables the calibration ofall channels in the flow cytometer with the same set of particles. Thefluorophores used are very stable but non-spectral matching to commonlyused fluorophores such as FITC, PE or PE-Cy5. Dilution of a few drops ofthe particles flam the chopper bottle to 1 mL of a diluent providesadequate particle concentration for flow cytometer calibration. Thediluted Rainbow Calibration Particles remain stable following repeatfreezing and thawing.

Strepatividin-Biotin Reaction in a Tubular Processor and a MicrofugeTube

A tubular processor for use as a diagnostic device preferably is able tocarry out an assay without loss in sensitivity. Moving the particlesthrough oil could denature the proteins bound to the particle or foam alayer on the particle making diffusion from the bulk solution to theparticle surface difficult.

A streptavidin-biotin system was utilized. The biotin-avidin orbiotin-streptavidin interaction has some unique characteristics thatmake it beneficial as a general bridge system. Avidin, streptavidin, andNeutrAvidin biotin-binding protein each bind four biotins per proteinmolecule with high affinity and selectivity.

Unlabeled Streptavidin coated SPHERO Smooth Surface Magnetic Particles(1% w/v) with an average diameter of 3μ were used for the experiment. Ineach of the following experiments, 0.7*10⁶ magnetic particles were used.The particles were washed in Phosphate Buffer Saline (PBS), magneticallyseparated and resuspended in 60 μL of PBS buffer containing 0.1% NonidetP-40 detergent. Different concentrations of Alexa-488-Biotin weredissolved in Na-Phosphate buffer containing 0.1% Nonidet P-40

The following solutions were injected into the capillary in the sequencegiven, each separated by 60 μL of silicone oil:

(a) 60 μL of PBS buffer containing the magnetic particles(b) 60 μL wash buffer of PBS containing 0.1% Nonidet P-40(c) 200 μL Na-Phosphate buffer containing a known concentration ofAlexa-488-Biotin and 0.1% Nonidet P-40(d) 60 μL wash buffer of PBS containing 0.1% Nonidet P-40

The particles were first moved magnetically from ‘solution-a’ to thechamber containing the wash buffer (solution-b). This cleans theparticles of debris. They were then moved to the chamber containing theAlexa-488-Biotin allowing the Streptavidin coated particles to bind toAlexa-488Biotin (solution-c). The particles were constantly mixedmagnetically. The reaction was allowed to continue for 90 minutes andthen the particles were moved to the next chamber (solution-d) to washoff any debris which might have stuck to the particles. Moving theparticles from one chamber to the next through the silicone oil involvesthe particles coming together into a clump so that they can cross theoil-water barrier. Hence, each time the particles move across from onechamber to the next, they are made to mix well by moving the magnets.The particles are then collected and fluorescence on the particle ismeasured in a flow cytometer.

The Alexa Fluor dyes used for the experiment are a series of superiorfluorescent dyes that span the near-UV, visible, and near-IR spectra.These dyes, without exception, produce brighter conjugates compared tofluorescein. The Alexa-488 absorbs light at 495 nm, emits at 519 nm andhas an extinction coefficient of 71000. The dye is water soluble andremains highly fluorescent over a broad pH range.

An identical reaction was carried out in a microfuge tube. 0.7*10⁶particles were washed in PBS, magnetically separated and resuspended in60 μL of PBS buffer containing 0.1% Nonidet P-40 detergent. Theparticles were then mixed with the following solutions each time beingmagnetically separated before being resuspended in the next solution.

(a) 60 μl of PBS buffer containing the magnetic particles(b) 60 μL wash buffer of PBS containing 0.1% Nonidet P-40(c) 200 μL Na-Phosphate buffer containing a known concentration ofAlexa-488-Biotin and 0.1% Nonidet P-40(d) 60 μL wash buffer of PBS containing 0.1% Nonidet P-40

As was the case of the reaction in the capillary, the Streptavidincoated magnetic particles were allowed to mix with biotin containingbuffer for 90 minutes. The particles were mixed constantly during thistime. Fluorescence readings were taken from these particles and comparedwith the fluorescence readings of the particles from the capillarysystem.

FIG. 8 shows the value of FL1 Height measured in a flow cytometer atdifferent concentrations of Biotin. The diamonds represent the signalfor a Streptavidin biotin reaction carried out in the capillary systemwhereas the pink squares represent the signal for a Streptavidin biotinreaction carried out in the microfuge rube. All measurements werestandardized by keeping the third peak of the rainbow at a fixed value.As can be seen from the plot, the particles are completely saturateduntil a Biotin concentration of 10⁻¹⁰ M after which the signal startsreducing. The amount of biotin which binds on to the Streptavidin coatedparticles is similar irrespective of whether the reaction is carried outin a capillary or a microfuge tube. The difference between the tworeactions is not statistically significant at all measuredconcentrations of biotin. The graph shows that there is some quenchingin the fluorescent signal at high concentrations of biotin. FIG. 8 shows5 replicates for the Streptavidin-biotin reaction in a tubular processorat a biotin concentration of 0.5*10⁻¹⁰M. This represents the variabilityof the reaction from one run to another.

The procedure followed for the experiment is the same as the previousexperiment described above. FIG. 9 consists of two superimposed imagesmeasured for two sets of particles, namely:

(a) Streptavidin coated SPHERO Smooth Surface Magnetic Particlesreacting with 10⁻¹²M Biotin solution(b) Streptavidin coated SPHERO Smooth Surface Magnetic Particlesreacting with deionized water

The forward scatter and the side scatter is the same for the two sets ofparticles, as would be expected because of identical size and surfaceroughness of the two sets of particles. The FL1 height, which is ameasure of the amount of fluorescence emitted off the particle surfaceand thereby a direct measure of the amount of biotin bound toStreptavidin is different for the two sets of particles. FIG. 9 showsthat the median FL1 height (peak) when the particles reacts with 10⁻¹²MBiotin is distinctly distinguishable and greater than the median heightwhen the Streptavidin coated particles react with water. The sensitivityof the device was found to be 10⁻¹² M for biotin (more than 2 dB abovethe blank).

Particle Transport Between Tubes

As shown in FIG. 10, one tube is inserted into another such that theouter diameter of the thinner tube is almost equal to the inner diameterof the larger tube. Streptavidin coated particles are moved to thechamber containing PBS buffer. While doing so, it also moves from onecapillary to another. It is then moved to a chamber containing PBSbuffer containing 10⁻⁸M Alexa-488-Biotin and 0.1% Nonidet-P40 (NP-40).Moving from one chamber to another involves crossing regions of siliconeoil. The particles were allowed to react with Alexa-488-biotin for 90minutes, after which the particles are taken out and their fluorescencemeasured in a flow cytometer. The fluorescent readings indicate that thereaction occur s in spite of moving from one capillary to another.

In a portable diagnostic device, the reagents might be potentiallypresent in one tube while the sample to be measured may be collected inanother a separate tube or vessel suitable for collection of sample.This experiment demonstrated the feasibility of allowing the particlesto react with the sample in a tube or collection vessel and then movingthem to another tube where the remaining reagents are present, therebyallowing the proceeding reactions to be carried out.

Effect of Silicone Oil

One of the factors that affects the diagnostic device is the effect ofthe silicone oil on the particles. It was investigated if the oil sticksto the particle during the movement of the particle through the oilregion. For this purpose, a fluorescent dye, pyromethene 546, wasdissolved in silicone oil. Pyromethene is an oil soluble laser dye whichfluoresces at 546 nm. The particles were made to move through a regionof silicone oil containing pyromethene 546. The particles were thentaken out of the capillary and the fluorescence signal was measured in aflow cytometer (FIG. 11 a).

In another experiment, the particles were moved through silicone oilcontaining pyromethene 546 and then mixed in PBS buffer containing 0.1%Nonidet P40. A magnet was used to agitate these particles. The particleswere then taken out of the capillary and the fluorescence signalmeasured in a flow cytometer as shown in FIG. 11 b.

The forward scatter value depicts the size of the particle while theside scatter gives information about the surface property of theparticle. In FIG. 11 a, the forward scatter plot shows a broaddistribution although the particles are about the same size. Thisdemonstrates that the oil is sticking to the particle or droplets of oilof various sizes are formed. Upon agitating the particles in PBS bufferafter moving the particles through oil, the particle size distributionshows two distinct peaks as expected, namely the particles and thedoublets of the particles (FIG. 11 b). The side scatter plot in FIG. 11a also shows two peaks suggesting that there might be little droplets ofoil formed as well. The FL1 median value of FIG. 11 a is 73 whichdemonstrates that some of the fluorescent dye has migrated with theparticles. This can be washed away by mixing the particles and thereforea lower fluorescence value 38 is obtained in FIG. 11 b.

Example 4 Efficiency of Commercial Viral RNA Purification Using LiquidWax

This example demonstrates that a purification method that transports RNAbound to paramagnetic particles through a liquid wax medium is adaptableto commercially-available kits which employ different types of particlesand a variety of lysis, wash, and elution buffers. The kits testedcontained silica (Ambion), iron oxide (Abbott/Promega) and cellulose(Cortex) magnetic particles. In addition to differences in particlechemistry, these kits vary in the composition of their respective lysisand elution buffers as well as the intermediate wash buffers.

Viral particles spiked into normal plasma were purified according to thekit manufacturers' instructions, including all of the wash steps betweenlysis and elution, and determined the levels of RNA by real time PCR.Spiked plasmas were then purified with the wax phase method using onlythe lysis and elution buffers and compared levels of RNA.

Comparison of RNA concentrations, expressed in Ct units, showed that theefficiency of the liquid wax transfer purification methodology isequivalent to that of procedures in manufacturer-specified guidelines.Taken together, these results indicate that the exclusion of the lysisbuffer by liquid wax is an appropriate replacement for the multiplemanual washes typically prescribed by RNA purification systems.

Wax-Bridge Cuvette

All experiments were performed in a two-chamber cuvette shown in FIGS.12 and 13, which were designed to facilitate moving particles from thelysis buffer to the elution buffer. As shown in FIG. 12, lysis buffer isadded to the chamber on the left, and elution buffer is added to thechamber on the right. Liquid wax is then added which covers both buffersolutions and forms a bridge between the two chambers. When a magnet isplaced on the side wall of the lysis chamber, particles are drawn to thewall forming a pellet. As shown in FIG. 13, the magnet is then moved updragging the pellet along the wall through the lysis buffer into the oillayer. The magnet is then moved laterally dragging the particles throughthe oil bridge until the pellet is above the elution chamber. Finally,the magnet is moved down dragging the particles out of the oil into theelution buffer.

Manufacturers' Protocols

The Ambion MagMax Viral RNA Isolation Kit(Catalog No. AM1929),Abbott/Promega M Sample Preparation System (Catalog No. 02K02-24), andCortex Biochem MagaZorb® RNA Isolation Kit(Catalog No. MB2001) weretested. Viral RNA samples were processed with each manufacturer'sreagents following the kit protocols, which include multiple washingsteps.

Liquid Wax Protocols

Ambion MagMax Viral RNA reagents:

Sample Lysis

25 μL of plasma containing 1.5×10⁶ cp/mL HIV-1 virions was added to 802μL of Lysis buffer (composition: 400 μL manufacturer-suppliedLysis/Binding concentrate, 400 μL absolute isopropanol, 2 μLmanufacturer-supplied carrier RNA) in 1.5 mL screw-cap tubes and mixedby pipet. Lysis proceeds for up to 10 minutes at 50° C., in the presenceof 20 μL well-suspended magnetic particles (manufacturer-supplied;composition: 10 μL particles, 10 μL Binding Enhancer). Particles may beadded following the initial lysis step or be present during lysis; nodiscernable effect on purification efficiency has been observed.

Cartridge Setup

Magnetic particles are sedimented on a magnetic rack and 600 μL ofsupernatant liquid is discarded to permit loading into a preparedcartridge. Up to 250 μL of the slurry containing lysis buffer andRNA-bound particles is transferred to the ‘lysis’ chamber of thecartridge. 25 μL, of a manufacturer-supplied Tris-EDTA elution buffer isloaded into the ‘elution’ chamber of the cartridge. Chill-Out Liquid Wax(Bio-Rad) is layered on top of the fluid in both chambers such that awax ‘bridge’ is formed across the top of the cartridge, typicallyrequiring 800 μL of liquid wax. Cartridges for multiple samples may beprepared in batch in this manner and arrayed on a fabricated rack.

Purification

RNA-bound beads are accumulated into a tight pellet by a magnet.Cartridges are handled individually for the transfer of particles fromlysis' to ‘elution’ chambers. Magnetic particles are transferred throughthe wax and into the elution buffer by magnet in a steady manner suchthat the beads remain in a tight pellet and carryover of lysis buffer isminimized. Once in the elution buffer, the beads are mixed by manualmanipulation of the magnet.

Sample Recovery

Liquid wax is aspirated from the cartridge such that a minimal amount (1mm at meniscus) remains over the ‘elution’ chamber without sample loss.The cartridge is set for 4 minutes at −20° C. such that the liquid waxsolidifies but the elution buffer/bead slurry remains in liquid phase.The liquid wax plug can be removed by pipet tip or pierced forextraction of elution buffer. The elution buffer/bead slurry istransferred to a 1.5 mL screw-cap tube and heated for up to 10 minutesat 70° C. to facilitate complete elution of viral RNA into thesupernatant buffer. Beads are sedimented on a magnetic rack and up to 50μL of RNA in Tris-EDTA buffer is collected from each tube, ready to usefor qRT-PCR.

Abbott/Promega Reagents: Sample Lysis

25 μL of plasma containing 1.5×10⁶ cp/mL HIV-1 virions was added to 600μL of Lysis buffer (supplemented with 2 μL manufacturer-supplied carrierRNA) and 25 μL iron-oxide magnetic particles in 1.5 mL screw-cap tubesand mixed by pipet. Lysis proceeds for up to 10 minutes at 50° C.

Cartridge Setup

After lysis, magnetic particles were sedimented and 400 μL ofsupernatant was discarded. 25 μL of a high-salt elution buffer is addedto the ‘elution’ chamber. Up to 250 μL, of the slurry containing lysisbuffer and RNA-bound particles is transferred to the ‘lysis’ chamber ofthe cartridge. Chill-Out Liquid Wax (Bio-Rad) is layered on top of thefluid in both chambers such that a wax ‘bridge’ is formed across the topof the cartridge, typically requiring 800 μL of liquid wax. Cartridgesfor multiple samples may be prepared in batch in this manner and arrayedon a fabricated rack.

Purification and Sample Recovery

A procedure identical to that employed for the Ambion kit was used.

Cortex Biochem Reagents: Sample Lysis

25 μL of plasma containing 1.5×10⁶ cp/mL HIV-1 virions was treated with20 μL Protease K by gentle mixing in a 1.5 mL screw-cap tube. 200 μL ofmanufacturer-supplied Lysis buffer was added and the sample mixed bypulse-vortexing for 15 seconds, followed by heating up to 15 minutes at55° C. 500 μL of manufacturer-supplied Binding buffer and 20 μL ofmonodisperse MagaZorb Reagent was added to the sample and incubated 10minutes at room temperature (20° C.) with occasional mixing byinversion.

Cartridge Setup

Magnetic particles are sedimented on a magnetic rack and 500 μL ofsupernatant liquid is discarded to permit loading into a preparedcartridge. Up to 250 μL of the slurry containing lysis buffer andRNA-bound particles is transferred to the ‘lysis’ chamber of thecartridge. 25 μL of a manufacturer-supplied Tris-EDTA buffer was addedto the ‘elution’ chamber. Chill-Out Liquid Wax (Bio-Rad) is layered ontop of the fluid in both chambers such that a wax ‘bridge’ is formedacross the top of the cartridge, typically requiring 800 μL of liquidwax. Cartridges for multiple samples may be prepared in batch in thismanner and arrayed on a fabricated rack.

Purification and Sample recovery

A procedure identical to that employed for the Ambion kit was used, withthe exception that the elution buffer/bead slurry was heated to 70° C.for 15 minutes to facilitate elution of viral RNA.

Comparison of Procedures

The adaptability of various viral RNA purification chemistries to theliquid wax purification method was evaluated by qRT-PCR using reagentssupplied with the Abbott m2000rt assay kit. In each case, HIV-1 virioncontaining plasma was used as template for qRT-PCR analysis and wascomplemented by appropriate positive (pure HIV-1 transcript) andnegative (mock plasma purification or water) controls. All purificationsamples include carrier RNA as an internal control and were performed inreplicate. From each RNA preparation, 5 μL RNA was used for analysis,yielding 7,500 copies of HIV-1 RNA in each reaction. A qRT-PCR mixturefor a single sample is described in Table 4 below:

TABLE 4 Component Volume (μL) m2000rt oligo. pre-mix 8.75 Tth polymerase(Roche) 0.75 Manganese acetate (Roche) 2.5 Crowding reagents (5X) 5.0Purified RNA 5.0 Nuclease-free dH₂O 3.0Results of qRT-PCR Analysis

Ct values given by the qRT-PCR instrumentation are given in Table 5below:

TABLE 5 Multi-step Liquid Wax Wash Magnetic particle type Mean Ct MeanCt Silica (Ambion) 20.69 18.69 Iron oxide 21.86 19.00 (Abbott/Promega)Cellulose (Cortex 20.32 19.83 Biochem)For a set number of HIV-1 template copies, there was little varianceobserved in the reported Ct values from qRT-PCR analysis betweenchemistries tested and purification method employed. Negativeamplification and mock purification controls in these experiments gaveno or negligible Ct values in these experiments. The phase gatepurification procedure is comparable in efficiency to kit manufacturerprotocols, demonstrating that the liquid wax exclusion of lysis bufferand other PCR inhibitors is as effective as multiple washing.Spectrophotometry of eluted RNA samples from the Abbott/Promega samplesshows no absorbance at 230 nm. Guanidinium is present in high molarconcentrations in this lysis buffer and absorbs ultraviolet light at 230nm; the absence of an absorbance peak shows that the carryover of thiscontaminant is low. That the observed Ct values are consistent acrossreplicates, largely invariant amongst purification chemistries andcomparable between purification methodologies demonstrates that good RNArecovery has been achieved.

Example 5 Isolation of CD4+ T-Cells

Fresh Peripheral Blood Mono-Nuclear Cells (PBMNC's) were purchased fromAllcells (Emeryville, Calif.). Dynabeads CD4 magnetic particles (4.5 umdiameter), coated with anti-CD4 antibody were purchased from Invitrogen(Carlsbad, Calif.).

1.2 ml of PBMNC's were mixed with 50 μL of the Dynabeads CD4 magneticparticles. The capture reaction was allowed to go for 45 minutes at 4°C. with gentle tilting and rotation. The CD4+ve T-Cell positiveisolation was compared using three procedures.

a) Positive isolation using Dynabeads CD4 protocol: 200 μL of the abovestock was aliquoted into the Well 1 of the research cartridge (shown inFIG. 12, labeled lysis buffer, and FIG. 13). A magnet was placed on theside of the cartridge for 1 minute, causing the magnetic particles andcaptured cells to form a pellet on the side. The supernatant wasaspirated and discarded. The pellet was washed 3 times by re-suspendingin 200 μl PBS and separating using a magnet. After the final wash, 60 μlof 0.2% Triton X-100 was added to lyse the cells.

b) Positive extraction through Chill-Out Liquid Wax (Bio-Rad, Hercules,Calif.). 200 μl of the stock solution was aliquoted into Well 1 of theresearch cartridge. 60 μl of 0.2% Triton X-100 lysis buffer wasaliquoted into Well 2 of the research cartridge (shown in FIG. 12,labeled elution buffer, and FIG. 13) which was separated from Well 1 byChill-Out Liquid Wax. A magnet was placed on the side of the cartridgefor 1 minute, drawing the magnetic particles and captured cells onto theside of Well 1. The pellet was then slowly moved through the wax intoWell 2 by dragging the magnet along the path shown in FIG. 13. Themagnet was used to agitate the particles in Well 2.

c) Positive extraction through canola oil (Jewel-Osco/Supervalu, EdenPrairie, Minn.). 200 μl of the stock solution was aliquoted into Well 1of the cartridge. 60 μl of 0.2% Triton X-100 lysis buffer was aliquotedinto the lysis chamber which was separated from Well 2 by canola oil. Amagnet was placed on the side of the cartridge for 1 minute, drawing themagnetic particles and captured cells onto the side of Well 1. Thepellet was then slowly moved with a magnet through the canola oil intoWell 2. The magnet was used to agitate the particles in Well 2.

The efficiency of the transfer through the lipophilc barriers wasdetermined by measuring the amount of cellular DNA in an aliquot of thesolution in Well 2 with a real-time PCR assay for the β2-microglobulingene. In each of the experiments, the particles were drawn to the sideof Well 2 with a magnet; and 60 ul of the lysed cell suspension wastransferred into Eppendorf tubes. 5 ul of each sample was then obtainedand used for RT-PCR using the Phusion GC assay for β2-microglobulin (NewEngland Biolabs, Ipswich, Mass.)

Results

Table 6 below shows the number of cycles required to reach the thresholdfluorescence intensity of the real time PCR assay:

TABLE 6 Cell purification Ct value DynalBeads protocol 27.59 Chill-OutWax 27.14 Vegetable Oil 28.99

The results show that the Ct values obtained from the three cellisolation procedures were comparable. Since the assay quantifiedβ2-microglobulin, the results show that the genetic material in thecells was preserved while being moved through the wax and oil.

Example 6 RNA Purification from Plasma Using Dextran PMPs

In order to eliminate all wash steps for purifying nucleic acids and toeliminate all contact between the processing system and the sample, PMPswere transported between wells using an externally applied magneticfield. The wells are connected with a hydrophobic liquid through whichPMPs are transported (FIG. 12). The hydrophobic liquid acts as a barrierbetween the lysis chamber and the elution chamber, preventing mixing ofthe two solutions. Upon application of the magnetic force, the PMPs aremoved through the hydrophobic liquid, transporting NAs from the lysischamber to the elution chamber while the lysis and elution buffersremain stationary. The hydrophobic liquid acts as an immiscible phasefilter (IPF), which reduces processing to only three steps: celllysis/NA binding, PMP transport, and NA elution. To demonstrate thefeasibility of incorporating IPF into a RNA purification protocol, HIV-1RNA was extracted from plasma as is done in measuring viral load.Quantitative measurement of HIV-1 is important for monitoring diseaseprogression and evaluating antiretroviral drug therapy outcome(Mylonakis, Paliou et al., Am. Fam. Phisician 63(3) 483 2001). Sinceviral load measurement is technically demanding due to the relativelylow viral copy number and abundance of PCR inhibitors in samples derivedfrom human blood (Dineva, Mahilum-Tapay et al. Analyst 132: 1193-11992007), this assay provides a good model system.

HIV-1 virus, acquired from Rush Virology Quality Assurance Laboratory at1.5×10⁶ copies/ml of plasma, was diluted in seronegative plasma toobtain HIV-1 concentrations of 300, 60 and 12 copies/μL respectively.The Ambion MagMax™ Total RNA isolation kit (Applied Biosystem; FosterCity, Calif.) manual protocol was performed as per manufacturer'srecommendations. For purification with the IPF method, lysis and bindingreagents constituting of 200 μL of Ambion Lysis/Binding solutionconcentrate (Applied Biosystem; Foster City, Calif.), 200 μL ofisopropyl alcohol, 1 μL of carrier RNA (Applied Biosystem; Foster City,Calif.), 5 μL of Ambion PMPs and 5 μL of Binding Enhancer (AppliedBiosystem; Foster City, Calif.) were added to the larger chamber of thecartridge and mixed. 50 μL of plasma containing HIV-1 virus was thenadded to it and mixed for 4 minutes using the automated system. 50 μL ofelution buffer was aliquoted into the smaller chamber of the IPFcartridge and the two aqueous fluids were overlaid with Chillout™ liquidwax (Biorad Laboratories; Hercules, Calif.) as shown in FIG. 12. Anautomated system aggregated the PMPs for 2 minutes using the externalmagnet and moved the aggregate from the lysis buffer to the elutionbuffer. The elution buffer containing the PMPs was heated to 55° C. for10 minutes to elute the RNA. The PMPs were aggregated and removed fromthe elution buffer. HIV-1 viral load quantification was performed usingthe Abbott RealTime HIV-1 Amplification Reagent Kit (Huang, Salituro etal. 2007) (Abbott Molecular, Des Plaines, Ill.) in 25 μl reactionvolumes with the addition of 0.2 mg/ml bovine serum albumin (B8667,Sigma), 150 mM trehalose (T9531; Sigma) and 0.2% Tween 20 (28320; PierceThermo Fisher Scientific) and 5 μl template. Amplification reactionswere performed in Cepheid SmartCycler II (Sunnyvale, Calif.).

The purified RNA was amplified using the Abbott RealTime HIV-1Amplification Kit. A PCR efficiency of E=102% was observed (FIG. 14),indicating that the inhibitor carryover is minimal even aftereliminating the four wash steps and the alcohol evaporation steprequired for the standard protocol. Comparing the IPF and the standardprotocol for RNA purification using the Ambion MagMax™ Total RNAisolation kit showed that at 0.05 level of significance (α=0.05), therewas no statistical difference between the two methods (p-value=0.967)(FIG. 15). Ten replicates each at several low copy numbers were purifiedand it was found that 60 copies of viral RNA could be detected in thePCR reaction with 100% sensitivity. Because 50 μl of plasma were used,this corresponds to 600 copies per ml of blood with 100% sensitivity.

Example 7 Purification of Chlamydia and Gonorrhea DNA from Urine

In order to eliminate all wash steps for purifying nucleic acids and toeliminate all contact between the processing system and the sample, PMPswere transported between wells using an externally applied magneticfield. The wells are connected with a hydrophobic liquid through whichPMPs are transported (FIG. 12). The hydrophobic liquid acts as a barrierbetween the lysis chamber and the elution chamber, preventing mixing ofthe two solutions. Upon application of the magnetic force, the PMPs aremoved through the hydrophobic liquid, transporting NAs from the lysischamber to the elution chamber while the lysis and elution buffersremain stationary. The hydrophobic liquid acts as an immiscible phasefilter (IPF), which reduces processing to only three steps: celllysis/NA binding, PMP transport, and NA elution. To demonstrate thefeasibility of using the IPF method to extract NA from urine, bacterialDNA was purified from Chlamydia trachomatis (CT) and Neisseriagonorrhoeae (NG) for diagnosis of these sexually transmitted diseases.

The urine samples were prepared by combining Chlamydia: ATCC trachomatisserotype F in McCoy cell culture suspension and lyophilized Neisseriagonorrhoeae resuspended in PBS containing 30% glycerol with controlurine (Fisher Scientific, Pa.). The manual protocol was carried outusing the Abbott RealTime CT/NG assay as per the manufacturer'sprotocols. For purification with IPF method, 200 μL of AmbionLysis/Binding solution concentrate (Applied Biosystem; Foster City,Calif.), 200 μL of isopropyl alcohol, 1 μL of carrier RNA (AppliedBiosystem; Foster City, Calif.), 5 μL of Ambion PMPs and 5 μL of BindingEnhancer (Applied Biosystem; Foster City, Calif.) were mixed. 200 μL ofurine sample was then added to it. The solution was heated to 55° C. for10 minutes and the two-step purification was carried out as with theplasma samples. As described previously (Marshall et al., J. Clin.Microbiol., 45:747-51, 2007), the purified DNA was amplified using theAbbott RealTime CT/NG assay in a 50 μL reaction volume. Amplificationreactions were performed in the Abbott Molecular m2000rt instrument(Abbott Park, Ill.).

The PCR efficiency for the CT and NG assay over seven orders ofmagnitude was 97.2% and 94.5% respectively (FIG. 16-17) indicating thatthe inhibitor carryover is minimal. These efficiencies were similar tothose obtained from the manual extraction method (87.9% and 87.9%,respectively) using the Abbott RealTime CT/NG kit. The Bland-Altmanplots of the CT and NG assays show that there is no statisticaldifference between the standard method using the Abbott DNA purificationkit and the IPF method. (FIG. 18-19). At a 0.05 level of significance(α=0.05), the two were found to be identical (p-values of 0.42 and 0.70respectively).

Example 8 Purification of Genomic DNA from Whole Blood

In order to eliminate all wash steps for purifying nucleic acids and toeliminate all contact between the processing system and the sample, PMPswere transported between wells using an externally applied magneticfield. The wells are connected with a hydrophobic liquid through whichPMPs are transported (FIG. 12). The hydrophobic liquid acts as a barrierbetween the lysis chamber and the elution chamber, preventing mixing ofthe two solutions. Upon application of the magnetic force, the PMPs aremoved through the hydrophobic liquid, transporting NAs from the lysischamber to the elution chamber while the lysis and elution buffersremain stationary. The hydrophobic liquid acts as an immiscible phasefilter (IPF), which reduces processing to only three steps: celllysis/NA binding, PMP transport, and NA elution.

Whole blood (WB) is a rich source of genomic DNA; however, it is anextremely complex medium containing numerous PCR inhibitors in highconcentrations. To determine if the method could process such samples, aqPCR assay was developed to detect proviral HIV-1 DNA integrated intoperipheral blood mononuclear cells. Proviral DNA detection is usedroutinely to diagnose infants with HIV-1 (Read and Committee onPediatric AIDS Pediatrics 120(6): e 1547-1562 2007). The PromegaMagnesil gDNA purification kit which consists of 10 steps (lysis, 7washes, drying and elution) was adapted for use with the IPF whichinvolved 3 steps (lysis, PMP transport through liquid wax, and elution).

Cultured 8E5 cells (Folks, Powell et al. J. Exp. Med. 164(1): 280-2901986) (Rush Virology Quality Assurance Laboratory, Chicago, Ill.)containing a single copy of the HIV-1 genome per cell added to WB from aseronegative donor was used to simulate infant blood for the proviralDNA assay. The cells were thawed, counted using a hemoctyometer,serially diluted in phosphate buffer saline (PBS) and added to WB from aseronegative donor at concentrations of 8000 cells/μl, 1600 cells/4 320cells/μl and 64 cells/μl. The Promega Magnesil gDNA purificationprotocol was carried out at the manufacturer's recommendations. In theIPF method, 25 μL blood was added to 60 μL lysis buffer, agitated for aminute and incubated for 4 minutes at room temperature. 44 μL of lysisbuffer and 6 μL of PMPs was added, agitated for a minute and incubatedfor 4 minutes. 15 μL of lysis buffer and 200 μL of alcohol wash bufferwere added to the solution and the IPF purification was carried out asbefore. The purified DNA was amplified using the Abbott RealTime HIV-1Amplification Reagent Kit (Abbott Molecular, Des Plaines, Ill.) (Huang,Salituro et al. 2007) in 25 μl reaction volume. Amplification reactionswere performed in Cepheid SmartCycler II (Sunnyvale, Calif.).

Serial dilutions over 4 orders of magnitude yielded a standard curvewith a slope of −3.15 and PCR efficiency of 108% (FIG. 20). TheBland-Altman plot of the proviral PCR assays showed that there was nostatistical difference between the standard method using the Promegapurification kit and the IPF method (FIG. 21). At a 0.05 level ofsignificance (α=0.05), the two methods were found to be identical(p-value=0.98).

All publications, patents, patent applications and sequences identifiedby accession numbers mentioned in the above specification are hereinincorporated by reference in their entirety. Although the invention hasbeen described in connection with specific embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Modifications and variations of the describedcompositions and methods of the invention that do not significantlychange the functional features of the compositions and methods describedherein are intended to be within the scope of the following claims.

1. A method for isolating a protein from a sample, comprising: placing asample comprising a protein in a device comprising a first chamber thatcontains a water-containing solution, a second chamber that contains awater-containing solution, and a water-immiscible substance disposedbetween the solutions in the first and second chambers to definestationary interfaces between the water-containing solution in eachchamber and the water-immiscible material; applying an external, appliedforce to the sample in the first chamber, the applied force capable ofinteracting with the protein or with a component associated with theprotein; and transferring, by relative movement of the applied forcewith respect to the first chamber, the protein from the solution infirst chamber, into and through the stationary interfaces and theimmiscible substance, and directly into the solution in the secondchamber, whereby the protein is isolated from the sample.
 2. The methodof claim 1, wherein placing comprises placing the sample in the deviceand then depositing the water immiscible substance between the first andsecond chambers.
 3. The method of claim 2, further comprising placing asolid carrier component in a chamber of the device, the carriercomponent capable of associating with the protein.
 4. The method ofclaim 3, wherein the solid carrier is at least one magnetic particle. 5.The method of claim 4, wherein applying an external, applied forcecomprises applying an external magnetic force.
 6. The method of claim 4,wherein transferring comprises transferring the at least one magneticparticle with the protein associated thereto into and through the waterimmiscible substance.
 7. The method of claim 1, wherein the protein isan antibody.
 8. The method of claim 7, wherein the antibody is a HIVantibody.
 9. The method of claim 1, wherein transferring comprisesmoving a sample transport component that generates the force.
 10. Themethod of claim 1, wherein a chamber in the device comprises magneticparticles.
 11. The method of claim 1, wherein the water-containingsolution is a wash solution.
 12. The method of claim 1, wherein thewater-containing solution is a water-alcohol solution.
 13. The method ofclaim 1, wherein placing the sample in the device wherein the secondchamber comprises an elution medium.
 14. The method of claim 1, whereinthe first and second chambers are within a plurality of chambers, andthe first chamber is preceded by one or more chambers in the pluralityof chambers, and said placing comprises placing the sample comprising aprotein in a chamber preceding the first chamber.
 15. The method ofclaim 14, wherein a chamber preceding the first chamber comprises alysis reagent.
 16. The method of claim 15, wherein the chambercomprising the lysis reagent additionally comprises magnetic particlesas the component to interact with the protein and with the appliedforce.
 17. The method of claim 14, wherein a second of the one or morechambers preceding the first chamber comprises a chamber filled with awash medium.
 18. The method of claim 17, wherein the second chambercomprises an elution medium.
 19. A method for extracting a protein froma sample, comprising: providing a device comprising a first chambercomprising magnetic particles and a lysis buffer, a second chambercomprising a water-containing solution, a third chamber comprising awater-containing solution, and a channel connecting the second chamberand the third chamber, the channel comprising a water-immisciblesubstance to define stationary interfaces between the water-containingsolutions and the water-immiscible substance; introducing a samplecomprising a protein into the first chamber; applying an external forceto the sample, the force capable of interacting with the magneticparticles and associated protein; and moving the magnetic particles andassociated protein from the first chamber to the second chamber, andthen across the stationary interface into the water-immiscible substancein the channel and then across the stationary interface into the thirdchamber, said moving by relative movement of the force with respect tothe device, whereby said moving extracts the protein from the sample.20. The method of claim 19, wherein the force is a magnetic field. 21.The method of claim 19, wherein said moving comprises moving anexternally applied force relative to the device thus causing thetransfer of the protein.
 22. The method of claim 21, wherein said movingis automated.
 23. The method of claim 19, wherein said providingcomprises providing a device comprising an additional chamber before thefirst chamber and an additional chamber downstream of the third chamber,wherein the additional chamber before the first chamber is separatedfrom the first chamber by an air gap.
 24. The method of claim 23,wherein said placing comprising placing the sample into the additionalchamber before the first chamber.
 25. The method of claim 19, whereinsaid depositing comprises depositing the water-immiscible material toform a layer of the water-immiscible material over the solution in thesecond chamber.
 26. The method of claim 19, wherein said depositingcomprises depositing the water-immiscible material to form a layer ofthe water-immiscible material over the solution in the third chamber.27. The method of claim 19, wherein said placing comprises placing thewater-containing solution in the second chamber in an amount to form apathway of water-containing solution between the first chamber and thesecond chamber.
 28. The method of claim 19, wherein during said movingof the magnetic particles and associated protein, the lysis buffer inthe first chamber and the water-containing solutions in the second andthird chambers remain stationary.
 29. The method of claim 19, whereinthe protein is an antibody.
 30. The method of claim 29, wherein theantibody is a HIV antibody.
 31. A device, comprising: a first chambercontaining a water-containing solution into which a sample comprising aprotein can be placed; a second chamber containing a water-containingsolution in communication with the first chamber; a water-immisciblematerial disposed between the first and second chambers to form acontiguous barrier between the solutions in the first and secondchambers and to define stationary interfaces of water-containingsolution—water-immiscible material between the water-containing solutionin each chamber and the water-immiscible material; and a solid phasecarrier component disposed in the first chamber, said carrier componentmovable to the second chamber by an externally, applied force, saidsolid phase carrier component capable of associating with the protein ofinterest; wherein upon application of the external force to the solidphase carrier component it associates with the force and positionalmovement of the force relative to the first chamber and the secondchamber effects transfer of the solid phase carrier component and theassociated protein in the first chamber, into and through the waterimmiscible material and directly into the solution in the secondchamber.